THE  LIBRARY 

OF 

THE  UNIVERSITY 
OF  CALIFORNIA 

LOS  ANGELES 


DIRECTIONS 

FOR 

LABORATORY  WORK 

IN 

PHYSIOLOGICAL  CHEMISTRY. 


BT 

HOLMES  C.  JACKSON,  PH.D., 

Professor  of  Physiology,  University  and  Bellevue  Hospital  Medical  College, 
New  York  City. 


SECOND  EDITION,  REWRITTEN  AND  ENLARGED. 
SECOND    THOUSAND. 


NEW  YORK: 

JOHN  WILEY  &  SONS. 

LONDON:  CHAPMAN  &  HALL,   LIMITED. 

1911 


Copyright,  1902,  1908, 

BT 

HOLMES  C.  JACKSON. 


•3 


PREFACE. 


THE  second  edition  of  this  Manual  appears  as  the  result 
of  a  thorough  revision  of  the  original  edition,  which  was  issued 
last  year ;  numerous  additions  to  the  subject-matter  have  also 
been  inserted. 

In  its  preparation  the  author  has  had  iiv  mind  to  provide 
a  guide  for  systematic  work  in  a  course  in  Physiological 
Chemistry  such  as  is  given  in  the  majority  of  the  American. 
Medical  Schools.  An  attempt  has  been  made  to  present,  in 
experimental  form,  all  of  the  essentials  of  the  subject.  At 
the  same  time,  the  experiments  are  simple  and  of  a  form 
which  would  not  overstep  the  possibilities  of  equipment  and 
time  available  in  the  average  laboratory  in  physiological  chem- 
istry. Those  facts  which  do  not  allow  of  simple  experimen- 
tal study  have  been  ignored.  The  text  preceding  the  experi- 
ments has  been  limited  to  an  explanation  merely  sufficient  for 
a  correct  understanding  and  performance  of  the  work  indi- 
cated, with  the  expectation  that  the  laboratory  course  will  be 
supplemented  by  lectures  showing  the  important  relationship 
of  the  pure  chemistry  of  the  subject  to  physiological  and 
pathological  processes. 

The  description  of  the  experiments  has  been  set  down  with 
considerable  detail  and,  it  is  hoped,  clearness,  since  the  suc- 

iii 


578896 


iv  PREFACE. 

cessful  termination  of  tests  and  experiments  depends  to  such 
a  large  extent  upon  the  exact  manner  in  which  they  are  con- 
ducted. 

Direct  explanation  of  the  results  of  the  experiments  is  pur- 
posely omitted,  with  the  end  in  view  that  the  student  might 
be  led  to  reason  these  out  for  himself. 

Further,  it  has  been  presumed  that  the  student  taking  this 
work  does  it  in  preparation  for  the  practice  of  medicine.  Par- 
ticular emphasis  has  therefore  been  laid  on  that  point,  espe- 
cially in  the  part  devoted  to  urine  analysis,  where  those  meth- 
ods have  been  presented  which  adapt  themselves  particularly 
to  clinical  work. 

In  conclusion  the  author  claims  little  originality  in  the 
experiments,  and  acknowledges  the  use  of  all  text-books  and 
manuals  which  were  available. 

H.  C.  J. 


TABLE  OF  CONTENTS. 


PAGE 

CARBOHYDRATES 1 

Pentoses 2 

Monosaccharides 3 

Disaccharides 5 

Polysaccharides 7 

FATS 9 

PROTEINS 15 

Simple  proteins 20 

Conj  ugate  proteins , 25 

Derived  proteins 27 

MUSCULAR  TISSUE 29 

Proteins 29 

Nitrogenous  extractives 30 

Non-nitrogenous  extractives 34 

BONE  36 

NERVOUS  TISSUE 38 

Lipoids 38 

Cerebrosides 40 

Cholesterols 41 

SALIVARY  DIGESTION 43 

GASTRIC  DIGESTION 46 

Peptic  proteolysis 51 

PANCREATIC  DIGESTION 56 

Tryptic  proteolysis 57 

Amylolysis 60 

Lipolysis 60 

Pancreatic  rennin 61 

INTESTINAL  PUTREFACTION 62 

BILE 67 

Conjugate  acids 68 

Pigments 69 

V 


vi  TABLE  OF  CONTENTS. 

PAOE 

BLOOD 72 

Blood  serum 73 

Blood  plasma 74 

Form  elements 75 

Spectroscopic  examination 77 

MILK 80 

Quantitative  separation 81 

URINE.  .          85 

Normal  constituents 88 

Quantitative  ueierininations 103 

Pathological  constituents 121 

SEDIMENTS 134 

APPENDIX 137 

INDEX 145 


LABORATORY  WORK 

IN 

PHYSIOLOGICAL  CHEMISTRY. 


THE  CARBOHYDRATES. 

The  term  carbohydrate  is  usually  considered  as  embrac- 
ing those  compounds  which  contain  the  elements  C,  H,  and  0, 
the  H  and  0  being  present  in  the  same  ratio  as  they  exist  in 
water.  This  definition,  strictly  followed,  would  include  in  the 
group  substances  such  as  lactic  acid,  acetic  acid,  and  inosit, 
which  are  obviously  not  carbohydrates.  The  carbohydrates 
may  be  conveniently  divided  into  three  groups;  namely, 
Monosaccharides  or  Glucoses,  Disaccharides  or  Saccharoses, 
and  Polysaccharides  or  Amyloses;  but  a  more  strictly  chem- 
ical classification  would  designate  them  according  to  the 
number  of  carbon  atoms  present  in  the  molecule,  the  prefix 
aldo-  or  keto-  denoting  to  what  general  class  of  compounds 
(aldehydes  or  ketones)  they  belong:  thus  trioses,  tetroses, 
pentoses,  hexoses,  heptoses,  etc.,  and  aldohexose,  ketopen- 
tose,  etc.  Of  the  greatest  physiological  importance  are  the 
pentoses,  hexoses,  hexobioses  (Disaccharides),  and  polyoses 
(Amyloses). 

Note  the  general  character  and  appearance  of  the  van- 


2     LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY 

OILS  specimens  of  carbohydrates  which  are  presented  for 
study. 

Test  for  the  constituent  elements  as  follows: 

(a)  Carbon. — 1.  Heat  cautiously  some  of  the  substance 
on  a  platinum  foil.  The  piece  will  char,  owing  to  the  sepa- 
ration of  the  carbon  in  the  substance.  Further  heating  ren- 
ders the  carbon  capable  of  combining  with  the  oxygen  of  the 
air,  with  the  result  that  the  former  passes  off  as  C02,  and 
in  the  case  of  carbohydrates,  where  combustion  is  com- 
plete, no  residue  is  obtained.  A  substance  containing  oxy- 
gen in  sufficient  quantities  to  form  C02  with  all  the  carbon 
present  will  not  carbonize. 

2.  Mix  thoroughly  some  of  the  dried  substance  with 
powdered  CuO  and  place  the  mixture  in  the  bottom  of  a  dry 
test-tube.  Upon  warming,  the  carbon  of  the  substance  is 
oxidized  by  the  oxygen  of  the  CuO  and  escapes  as  C02. 
This  C02  may  be  detected  by  holding  a  glass  rod  moistened 
with  lead  acetate  at  the  mouth  of  the  test-tube. 

(6)  Hydrogen. — 1.  In  the  latter  experiment,  moisture  will 
have  collected  on  the  cold  part  of  the  test-tube.  The  hydro- 
gen of  the  substance,  in  the  presence  of  heat,  has  combined 
with  the  oxygen  supplied  by  the  CuO,  forming  H20. 

PENTOSES,  C5H1005. 
ARABINOSE.    XYLOSE. 

The  pentoses  do  not  occur  free  in  nature,  but  exist  in  the 
form  of  pentosanes — bodies  which  are  found  in  the  fruits  and 
polysaccharide  gums  (e.g.,  gum  arabic,  cherry  gum)  and  from 
which  the  pentoses  may  be  obtained  by  hydrolysis  with 
weak  acids.  The  ingestion  of  pentosanes  also  causes  pento- 
ses to  appear  in  the  urine.  Chemically  they  are  aldehydes 
and  as  such  reduce  Fehling's  solution.  With  regard  to  their 


THE  CARBOHYDRATES.  3 

action  on  polarized  light,  they  exist  in  three  modifications, 
but  those  derived  from  the  pentosanes  are  dextrogyrate. 
Pentose  solutions  when  heated  with  phloroglucinol  or  orcinol 
and  HC1  (sp.  gr.  1.09)  acquire  respectively  a  cherry- red  or 
green  color  and  posses  characteristic  absorption  spectra. 
The  pentoses  are  non-fermentable  and  yield  with  phenyl- 
hydrazin  osazones  having  characteristic  properties.  Boil 
some  cherry  gum  for  some  hours  with  1  per  cent  H2S04. 
Test  the  solution  for  pentose  as  follows: 

(a)  Saturates  c.c.  of  HC1  (sp.  gr.  1.09)  with  phloroglucinol 
by  warming  on  the  boiling-water  bath.  A  slight  excess  of 
the  phloroglucinol  is  advantageous.  Add  to  this  a  few  c.c. 
of  the  pentose  solution  and  continue  the  heating.  Grad- 
ually a  cherry-red  color  develops  in  the  solution  and  the 
latter  shows  characteristic  absorption  bands  between  D 
and  E. 

(6)  Try  the  same  test  using  orcinol  instead  of  phloro- 
glucinol. The  solution  turns  reddish,  then  reddish  blue,  and 
finally  green,  with  a  precipitate  of  that  color  settling  out  of 
the  solution.  This  precipitate  is  soluble  in  amyl  alcohol,  in 
which  solvent  it  also  has  characteristic  absorption  bands 
between  C  and  D. 

(c)  Prepare  pentosazones  after  the  method  employed 
on  p.  5. 

HEXOSES  OR  MONOSACCHARIDES,  C6H1206. 
DEXTROSE.        LEVULOSE.        GALACTOSE. 

The  monosaccharides,  in  general,  are  soluble,  crystalline, 
and  optically  active  bodies  which  yield,  upon  boiling  with 
alkalies,  oxidation  products  and  caramel;  they  reduce  metal- 
lic oxides  in  alkaline  solution;  ferment  with  yeast  and  with 
bacterium  lactis  ;  and  give,  with  phenylhydrazin,  osazones 


4     LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

with  characteristic  crystalline  forms,  solubilities,  and  melt- 
ing-points. 

GLUCOSE = DEXTROSE = GRAPE-SUGAR. 

Of  the  monosaccharides,  dextrose  holds  first  place  in 
importance  in  the  animal  economy.  Examine  and  taste  the 
dry  substance.  Test  its  solubility  in  water  and  in  hot  and 
cold  alcohol.  In  the  following  tests  make  use  of  a  1  per  cent 
solution: 

(a)  Moore's  Test. — To  5  c.c.  of  the  dextrose  solution  add 
an  equal  volume  of  NaOH,  and  heat.  The  mixture  becomes 
yellow  and  finally  brown,  due  to  the  formation  of  caramel. 
This  test  lacks  delicacy  and  reliability  in  examining  urine. 

(6)  Trommer's  Test. — To  5  c.c.  of  the  dextrose  solution 
add  an  equal  volume  of  NaOH.  Then  add,  drop  by  drop,  a 
dilute  solution  of  CuS04  (so  dilute  that  the  green  color  is  just 
visible)  until  a  trace  of  permanent  precipitate  remains.  The 
solution  should  be  deep  blue  in  color.  Warm  the  upper  part 
of  the  solution  and  note  result.  Write  all  the  equations  for 
the  reactions  taking  place  in  this  experiment. 

(c)  Fehling's  Test. — Heat  5  c.c.  of  Fehling's  solution  just 
to  boiling  and  add  a  few  drops  of  the  dextrose  solution.    Con- 
tinue the  boiling  until  the  solution  commences  to  respond  as 
with  Trommer's  test.     Compare  the  reactions  of  this  test 
with  those  of  the  previous  ones. 

(d)  Barfoed's  Test. — To  5  c.c.  of  Barfoed's  reagent  add  a 
few  drops  of  the  dextrose  solution  and  boil.     Note  the  result 
and  write  equations. 

(e)  Nylander's  Test. — To  5  c.c.  of  the  dextrose  solution  add 
10  drops  of  Nylander's  reagent  and  boil.     The  solution  grad- 
ually turns  yellow  and  finally  black,  bismuth  being  precipi- 
tated. 


THE  CARBOHYDRATES.  5 

(/)  Silver  Nitrate. — To  5  c.c.  of  AgN03  solution  -in  a  clean 
test-tube  add  dilute  NH4OH,  drop  by  drop,  until  the  precipi- 
tate, which  is  at  first  formed,  dissolves.  Then  add  a  few 
drops  of  the  dextrose  solution  and  warm  on  the  water-bath. 
Note  the  formation  of  a  metallic  mirror  on  the  side  of  the 
test-tube.  Explain  the  chemical  changes. 

(g)  Phenylhydrazin. — In  a  test-tube  prepare  a  mixture 
of  5  drops  of  phenylhydrazin,  10  drops  of  glacial  acetic  acid, 
and  1  c.c.  of  a  saturated  solution  of  NaCl;  then  add  5  c.c.  of 
the  dextrose  solution  and  boil  for  a  few  minutes.  Yellow 
phenylglucosazone  crystals  will  appear  on  cooling. 

Under  the  microscope  these  appear  as  fine  yellow  needles, 
usually  arranged  in  sheath-shaped  bundles.  If  determina- 
tions are  made  of  the  composition  and  melting-point  of  these 
crystals,  this  test  furnishes  most  characteristic  and  conclusive 
evidence  for  dextrose.  Write  the  equations  for  the  forma- 
tion of  the  osazones. 

(h)  Fermentation. — In  a  test-tube  shake  20  c.c.  of  the 
dextrose  solution  with  a  small  piece  of  compressed  yeast. 
Place  the  mixture  in  a  saccharometer  and  allow  it  to  stand 
in  a  thermostat  at  40°  C.  A  gas  collects  at  the  top  of  the 
tube  and  its  volume  is  in  a  direct  ratio  to  the  amount  of  dex- 
trose in  the  solution.  What  is  the  character  of  the  chemical 
changes  which  have  taken  place? 

(i)  Polarization. — See  demonstration  and  make  a  read- 
ing on  the  instrument  of  the  degree  of  right-handed  rotation. 

HEXABIOSES  OR  DISACCHARIDES,  C12H22On. 
SACCHAROSE.        LACTOSE.        MALTOSE. 

The  disaccharides  possess,  with  the  exception  of  saccha- 
rose, the  same  general  properties  as  the  simple  sugars.  As 
their  name  implies,  upon  hydrolysis  they  take  up  a  molecule 


6     LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

of  water  and  break  down  into  two  molecules  of  a  monosac- 
charide. 

This  decomposition  in  the  case  of  saccharose  has  assumed 
the  name  of  inversion  from  the  fact  that  when  a  dextro- 
rotatory solution  of  cane-sugar  suffers  hydrolysis,  the  result- 
ant mixture  is  Isevogyrate.  This  is  caused  by  the  strong 
laevo-rotation  of  the  Isevulose  more  than  overcoming  the 
dextrogyrate  polarization  of  the  dextrose. 

Saccharose  =  dextrose  +lsevulose. 
SACCHAROSE = CANE-SUGAR = SUCROSE. 

Sa3charose  differs  from  the  other  members  of  this  group 
in  not  reducing  metallic  oxides  in  alkaline  solution  and  in 
not  forming  osazones  with  phenylhyclrazin.  It  is  not 
directly  fermentable  with  yeast,  but  only  after  previous 
inversion  by  the  ferment  invertin,  secreted  by  the  yeast-cell. 
A  slight  reduction  which  is  sometimes  obtainable  with 
Fehling's  test  may  be  explained  by  the  inverting  action  of 
the  strong  alkali. 

Use  a  1  per  cent  solution  of  cane-sugar. 

Tests:  (a)  Fehling's;  Moore's;  Nylander's;  Barfoed's; 
using  in  each  case  5  c.c.  of  the  saccharose  solution.  Com- 
pare these  results  with  those  obtained  for  dextrose. 

(6)  To  10  c.c.  of  the  saccharose  solution  add  1  c.c.  concen- 
trated HC1  and  boil  several  minutes.  Allow  this  to  cool 
and  then  neutralize  with  NaOH.  Use  this  solution  in  making 
Fehling's,  Nylander's,  and  Barfoed's  tests.  Write  the  equa- 
tions for  the  chemical  changes  which  have  taken  place  and 
determine  the  character  of  the  carbohydrate  formed. 

Inversion  and  Fermentation. 

1.  Examine  the  saccharose  solution  in  the  polariscope 
and  determine  the  degree  of  rotation. 


THE  CARBOHYDRATES.  7 

2.  Place  10  c.c.  of  the  saccharose  solution  in  a  test-tube 
with  5  c.c.  of  1  per  cent  HC1.     Allow  the  mixture  to  remain 
two  hours  at  37°  C.     Finally  examine  this  in  the  polariscope 
and  test  its  reducing  power.     To  what  condition  in  the  body 
is  this  comparable? 

3.  To  10  c.c.  of  the  saccharose  solution  add  some  invertin 
and  allow  the  test-tube  to  remain  at  37°  C.    Test  for  invert 
action. 

4.  See  demonstration  of  the  formation  of  alcohol  and 
C02  from  a  fermentation  of  cane-sugar  by  yeast.     The  C02 
as  it  forms  is  caught  in  passing  out  of  the  flask  by  a  valve  of 
Ba(OH)2.     The  alcohol  is  distilled  off  from  the  mixture  in 
the  flask. 

MALTOSE  will  be  studied  under  Salivary  Digestion  and 
LACTOSE  under  Milk. 


POLYOSES  OR  POLYSACCHARIDES,  (C6H1005)«- 

STARCHES.      DEXTRINS.      GLYCOGEN.      CELLULOSES. 
VEGETABLE  GUMS. 

The  polysaccharides,  as  a  class,  are  amorphous,  more  or 
less  insoluble  substances  which  do  not  diffuse  through  animal 
membranes.  They  are  optically  active,  and  with  the  excep- 
tion of  the  dextrins  do  not  reduce  Fehling's  solution.  By 
hydration  with  weak  acids  or  with  enzymes  the  polysac- 
charides are  converted  into  monosaccharides ;  dextrins  and 
disaccharides  being  the  intermediate  products.  The  polysac- 
charides are  incapable  of  undergoing  fermentation  unless 
previously  inverted. 

STARCHES. 

The  starches  are  insoluble  bodies  which,  with  hot  water, 
form  opalescent  colloidal  solutions  or  pastes.  These,  if  suffi- 


8     LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

ciently  concentrated,  gelatinize  upon  cooling.  The  most 
characteristic  reaction  of  the  starches  is  their  behavior  toward 
iodine,  with  which  they  form  blue  compounds  whose  color 
disappears  upon  heating,  but  returns  as  the  solution  cools. 

(a)  Examine  under  the  microscope  and  sketch  the  follow- 
ing starch  granules:  potato,  corn,  wheat,  rice,  and  arrowroot. 

(6)  Place  some  starch  in  a  test-tube  half  full  of  water  and 
shake  thoroughly  until  tho  starch  is  finely  divided.  Heat 
water  to  boiling  in  another  test-tube  and  to  this  add  enough 
of  the  cold  starch  mixture  to  make  a  translucent  solution 
(about  2  per  cent).  What  takes  place  upon  pouring  the 
suspended  starch  into  the  hot  water?  The  solution  of  starch 
thus  obtained  is  called  a  paste,  and  in  making  experiments 
with  this  substance  such  a  paste  must  always  be  prepared. 

(c)  To  5  c.c.  of  starch  paste  add  a  drop  of  iodine  solution. 
Warm  gradually  and  then  allow  to  cool.     Note  changes. 

(d)  Boil  10  c.c.  of  starch  paste  with  1  c.c.  of  concentrated 
HC1  for  5  minutes.     Observe  the  change  which  the  solution 
undergoes.     Neutralize  part  of  this  cold  solution  with  NaOH, 
and  apply  Fehling's  and  Barfoed's  tests.     (If  no  reduction 
appears,  continue  the  boiling  of  the  original  acid  solution  for 
some  minutes  longer  and  repeat  the  neutralization  and  tests.) 
Determine  the  character  of  the  sugar  causing  this  reduction. 

For  the  DEXTRINS  see  Salivary  Digestion;  for  GLYCOGEN 
see  Muscular  Tissue. 


THE  FATS. 

The  neutral  fats  are  glycerol  esters  of  fatty  acids,  in  that 
three  hydrogens  of  the  carboxyl  groups  of  three  fatty  acids 
are  replaced  by  the  glycerol  radical.  A  general  formula  for 
a  neutral  fat  is  represented  thus: 

CH2-0-OC-R 
CH-0-OC-R' 


CH2-0-OC-R" 


in  which  R,  R',  and  R"  stand  for  three  of  the  same  or 
different  hydrocarbon  radicals.  In  the  molecule  of  the 
ordinary  animal  fats  R,  R',  and  R"  may  be  represented 
by  three  hydrocarbon  residues  of  palmitic,  of  stearic,  of 
oleic,  or  of  butyric  acid,  the  compounds  being  designated 
respectively  as  tripalmitin,  tristearin,  etc. 

The  specific  character  of  a  fat  is  dependent  upon  the  nature 
of  the  fatty  acid  in  the  molecule;  and  since,  in  general,  the 
melting-points  of  the  saturated  fatty  acids  increase  with 
their  carbon  content,  the  lower  compounds  remain  liquid  at 
ordinary  temperature  and  the  character  of  tributyrin  (R, 
R',  and  R" =C3H7)  is  that  of  a  soft  fat,  while  tristearin  (R,  R', 
and  R"  =  CiyH35)  possesses  considerable  firmness. 

Observe  the  olive  oil,  butter,  mutton  and  beef  fats  pre- 
sented for  study.  Pure  neutral  fats  are  devoid  of  color, 
odor,  and  taste,  and  their  low  specific  gravity  and  insolubility 


10  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

allow  them  to  float  on  the  surface  of  water.  They  are  solu- 
ble in  ether,  chloroform,  and  benzol;  hot  alcohol  also  dis- 
solves them,  but  upon  cooling  they  separate  out  usually  in 
crystalline  form.  Under  certain  conditions  fats  undergo  a 
peculiar  physical  change  called  emulsification,  which  is  beau- 
tifully illustrated  in  the  natural  state  of  the  fat  in  milk. 
Under  the  influence  of  superheated  steam,  acids,  or  ferments, 
fats  are  broken  down  into  their  component  parts — fatty 
acids  and  glycerol.  When  an  alkali  or  sodium  alcoholate  is 
employed,  soaps  and  glycerol  are  formed  and  the  process 
is  called  saponification.  Another  form  of  decomposition  is 
effected  when  fats  remain  for  any  length  of  time  in  contact 
with  the  oxygen  of  the  air.  They  then  become  rancid  by 
the  liberation  and  subsequent  oxidation  of  the  fatty  acid 
from  the  molecule  and  the  formation  of  lower  volatile  acids 
which  cause  unpleasant  odors  and  tastes. 

Try  the  following  tests,  making  use  of  olive  oil : 
(a)  Test  its  solubility  in  water;  ether;  chloroform;  alco- 
hol. 

(6)  Test  its  reaction.  What  is  the  normal  reaction  of  a 
fat?  Express  by  equations  what  occurs  when  butter  becomes 
rancid. 

(c)  In  a  test-tube  warm  a  few  drops  with  potassium  bisul- 
phate.     Notice  odor.     What  is  the  reaction  taking  place? 

(d)  Let  a  drop  of  an  ether  solution  of  a  fat  fall  upon  paper. 

(e)  Dissolve  a  little  lard  in  10  c.c.  of  a  mixture  of  equal 
parts  of  alcohol  and  ether.     Allow  this  to  remain  uncovered 
until  crystals  begin  to  form. 

(/)  Saponification  (Bayberry  Wax — Tripalmitin). 

Place  a  piece  of  the  wax,  half  the  size  of  a  walnut,  in  an 
evaporating-dish  which  is  half  full  of  water.  Then  add  about 


THE  FATS.  11 

10  c.c.  NaOH  and  boil  until  the  substance  is  dissolved,  adding 
water  from  time  to  time  as  the  solution  evaporates,  so  that 
the  original  volume  is  constantly  maintained.  Write  the 
reaction  which  is  taking  place.  After  complete  solution,  add 
carefully  dilute  H,S04  until  the  reaction  just  becomes  acid  and 
then  cool.  A  greenish  crust  of  free  fatty  acid  will  form  on 
the  surface  of  the  liquid,  so  that  the  solution  may  be  poured 
off.  (Keep  this  solution.)  Break  the  fatty  acid  crust  into 
small  pieces  and  wash  with  tap  water;  finally  dry  between 
filter-paper.  Dissolve  part  of  the  fatty  acid  in  50  c.c.  of  hot 
95  per  cent  alcohol,  filter  hot  through  a  dry  funnel  and  allow 
the  filtrate  to  cool  slowly. 

Palmitic  acid  separates  out  in  snowy-white  crystalline 
form.  Examine  some  under  the  microscope  and  sketch  the 
crystals. 

(g)  Saponification  (Lard), 

Place  about  100  c.c.  of  alcoholic  potash  in  a  flask  contain- 
ing 25  grams  of  lard.  Warm  the  mixture  upor.  the  water- 
bath  until  a  drop  let  fall  into  water  is  perfectly  soluble. 
Then  pour  the  solution  into  an  evaporating-dish  containing 
100  c.c.  water  and  evaporate  until  the  alcohol  has  been  driven 
off.  While  still  hot  acidify  with  dilute  H2S04  and  then 
cool.  Write  the  chemical  equations  for  the  various  steps  in 
the  procedure.  The  fatty  acid  rises  to  the  surface  and  may 
be  easily  removed  from  the  liquid  beneath.  Unite  this  solu- 
tion with  the  similar  one  in  Experiment  1  and,  after  neutrali- 
zation with  Na2C03,  evaporate  it  down  to  about  5  c.c.  What 
substances  are  present  in  this  solution?  Retain  this. 


12  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 


FATTY  ACIDS  AND  SOAPS. 

Since  the  character  of  the  fatty  acid  determines  the 
character  of  the  fat,  the  properties  and  solubilities  of  the 
fatty  acids  and  fats  must  be  similar,  as  the  tests  will 
prove. 

The  soaps  are  salts  of  the  fatty  acids;  the  sodium  salts 
being  solid  and  hard,  while  those  of  potassium  are  soft. 
Both  are  soluble  in  water.  The  remaining  metallic  salts 
are  insoluble  in  water,  but  generally  dissolve  in  alcohol. 
The  lead  combination  with  oleic  acid  (lead  plaster)  differs 
from  the  corresponding  one  with  palmitic  and  stearic  acids 
in  being  soluble  in  ether. 

Perform  the  following  reactions  with  the  fatty  (oleic)  acid: 

(a)  Test  its  solubility  in  ether;  chloroform;  water; 
alcohol. 

(6)  Warm  some  in  a  dry  test-tube  with  potassium  bisul- 
phate. 

(c)  Shake  up  a  few  pieces  of  the  fatty  acid  with  25  c.c. 
water  and  add  NaOH,  drop  by  drop,  until  the  acid  is  all  dis- 
solved. Write  the  reaction.  What  is  formed? 

Use  this  soap  solution  for  the  following  reactions: 

(a)  Add  5  drops  to  a  saturated  solution  of  NaCl. 

(6)  To  5  c.c.  of  lead  acetate  add  a  few  c.c.  of  the  soap 
solution.  A  sticky  precipitate  results  (lead  oleate),  which  is 
soluble  in  ether.  Write  the  reaction.  What  is  the  impor- 
tance of  this  compound? 

(c)  To  50  c.c.  of  tap  water  in  a  flask  add  the  soap  solu- 
tion, drop  by  drop,  alternately  shaking  until  a  permanent 
lather  is  obtained.  The  amount  of  soap  solution  required 
to  obtain  the  lather  is  in  direct  proportion  to  the  hardness  of 
the  water.  Explain  the  chemistry  of  this  experiment. 


THE  FATS.  13 


Emulsification  of  Fats. 

Certain  poorly  understood  factors  enter  into  the  forma- 
tion and  preservation  of  an  emulsion.  Apparently  the 
viscosity  of  the  menstruum  and  the  condition  of  the 
surface  tension  existing  between  that  menstruum  and  the 
globules  form  the  fundamental  physical  requisites  for  the 
obtaining  of  a  permanent  emulsion.  Solutions  of  gums, 
proteins,  and  soaps  emulsify  fats  with  varying  degrees  of 
permanency.  The  mere  presence  of  a  soap  in  the  solution  does 
not  seem  to  render  the  fat  particularly  prone  to  emulsify; 
the  necessary  condition  is  apparently  the  intermolecular  forma- 
tion of  a  soap  in  the  solution  as  exemplified  upon  the  addi- 
tion of  an  alkali  to  fat  containing  some  free  fatty  acid.  An 
emulsion  formed  in  this  manner  is  the  most  permanent  of  all 
those  induced  by  artificial  means;  a  similar  procedure  takes 
place  in  the  emulsification  of  fat  in  the  intestine. 

Make  up  the  following  mixtures  in  test-tubes: 

(a)  2  drops  of  neutral  olive  oil  +  10  c.c.  water. 

(6)  2  drops  of  neutral  olive  oil  +  10  c.c.  water  +  a  few 
drops  of  Na2C03. 

(c)  2  drops  of  neutral  olive  oil  +  10  c.c.  of  the  soap  solu- 
tion. 

(d)  2  drops  of  neutral  olive  oil  +  10  c.c.  of  an  egg-albumin 
solution. 

(e}  2  drops  of  rancid  olive  oil  +  10  c.c.  of  water  +  a 
few  drops  of  Na-jCOg. 

Shake  each  tube  approximately  the  same  length  of  time 
and  compare  the  relative  permanence  of  the  various  emul- 
sions. Which  conditions  are  the  most  favorable,  and 
why? 


14  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

CH2OH 

GLYCEROL,  CHOH 
CH2OH. 

Make  use  of  the  solution  obtained  from  the  two  saponifica- 
tions  for  the  following  tests : 

(a)  Note  the  taste  and  try  its  solubility  in  alcohol;  ether; 
water. 

(6)  Add  some  dry  potassium  bisulphate  and  warm. 
Compare  this  with  the  same  reaction  under  Fats.  Write  the 
equation. 

(c)  Test  with  Fehling's  solution. 

(d)  To  some  dilute  CuS04  add  the  remainder  of  the  glycerol 
solution;  then  drop  by  drop  add  dilute  NaOH.     What  causes 
the  blue  color  of  the  solution? 


THE   PROTEINS. 

The  proteins  are  complex  compounds  of  C,  H,  0,  N,  and  S 
(in  some  cases  P  and  Fe)  occurring  widely  distributed  in  the 
plant  and  animal  kingdoms.  The  members  of  this  class  of 
bodies,  although  differing  greatly  in  chemical  and  physical 
characteristics,  possess  in  common  certain  definite  properties 
and  chemical  reactions  which  allow  of  classification  and  sub- 
division. The  proteins  form  the  chief  type  of  the  food- 
stuffs, since  the  nitrogen  present  in  them  is  absolutely  essen- 
tial in  order  to  sustain  life. 

They  may  be  divided  as  follows: 

The  simple  proteins,  those  giving  upon  decomposition 
indole,  skatole,  tyrosine,  cystine,  amino  acids,  pyrrol  deri- 
vatives, etc. 

The  conjugate  proteins,  composed  of  a  simple  protein  and 
a  complex  non-protein  component. 

The  derived  proteins,  derivatives  of  the  proteins  and 
formed  through  hydrolytic  changes. 

Reactions  General  to  all  Proteins. 

For  the  following  tests  make  use  of  dried  egg-white  or 
casein : 

Test  the  substance  for  carbon  and  hydrogen. 

Nitrogen. — (a)  Make  an  intimate  mixture  of  the  sub- 
stance with  soda-lime  and  place  it  in  a  dry  test-tube.  Warm 
gently.  Hold  a  piece  of  moistened  red  litmus  paper  or  a 

15 


16  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

glass  rod  moistened  with  HC1  at  the  mouth  of  the  test-tube. 
What  is  the  reaction  which  takes  place? 

(6)  Warm  together  carefully  in  a  dry  test-tube  a  few 
particles  of  the  dry  substance  and  a  3-4  mm.  cube  of  metallic 
sodium.  (Caution:  Do  not  place  the  tube  in  the  flame. 
Gases  are  evolved  which  are  explosive  unless  the  temperature 
is  kept  moderate.)  When  the  fusion'  is  complete  and  the 
tube  has  cooled  somewhat  plunge  the  end  into  a  small  amount 
of  water  placed  in  a  suitable  vessel,  preferably  a  conical  glass. 
The  glass  of  the  test-tube  will  probably  break,  and  unless  the 
sodium  has  been  completely  fused  a  slight  explosion  will 
result.  When  the  water  has  thoroughly  impregnated  the 
fused  mass,  filter  and  to  the  filtrate  add  a  few  drops  of  ferric 
chloride  and  of  ferrous  sulphate  solution.  Upon  acidifying 
with  HC1  a  blue  precipitate  of  Prussian  blue  is  obtained. 
Write  all  the  reactions  which  take  place  in  this  manipulation. 

Sulphur. — The  sulphur  in  the  protein  molecule  exists  in 
part  in  such  a  form  that  upon  boiling  with  caustic  soda  it  is 
easily  split  off  as  sodium  sulphide.  This  in  the  presence  of 
a  lead  salt  forms  black  lead  sulphide.  On  this  account  such 
sulphur  has  assumed  the  name  of  loosely  combined  or  lead- 
blackening  sulphur.  If  the  quantity  of  sulphur  obtained  in 
the  above  manner  be  multiplied  by  |,  the  product  is  the 
amount  of  sulphur  considered  to  be  present  in  the  cystine 
nuclei  of  the  given  protein.  In  some  proteins — keratin, 
serum  albumin,  serum  globulin — the  total  sulphur  and  cystine 
sulphur  content  are  the  same  and  we  are  justified  in  assum- 
ing that  all  the  sulphur  in  the  molecule  exists  in  the  form  of 
cystine.  In  other  proteins  only  £— |  of  the  total  sulphur  is  to 
be  accounted  for  as  cystine  S;  hence  it  must  be  concluded 
that  sulphur  exists  in  some  atomic  complex  which  yields,  upon 
the  decomposition  of  the  molecule,  substances  like  a-thio- 
lactic  acid,  mercaptans,  ethyl  sulphide,  etc.  Sulphur  of  this 
type  may  be  styled,  in  contradistinction,  -firmly  combined 
sulphur.  The  sum  of  the  loosely  and  firmly  combined  sul- 
phur equals  the  total  sulphur  of  the  substance. 


WHE  PROTEINS.  17 

Test  for  lead-blackening  sulphur  as  follows: 

Place  about  5  c.c.  of  dilute  NaOH  in  a  test-tube  with  a 
small  quantity  of  the  substance,  and  add  two  drops  of  lead 
acetate.  Boil  the  mixture  for  a  few  minutes  and  note  the 
changes.  The  depth  of  color  may  be  said  to  correspond 
roughly  to  the  amount  of  neutral  sulphur  present  in  the  sub- 
stance. 

Test  for  total  sulphur  as  follows : 

Mix  some  of  the  substance  with  double  its  quantity  of  the 
fusion  mixture  (Na2CO3  +  KN03).  Place  the  whole  in  a 
small  porcelain  crucible  and  warm  cautiously  until  the  mix- 
ture becomes  colorless.  (If  the  fusion  begins  to  sputter, 
remove  the  flame.)  The  residue  (fused  mass)  should  be 
nearly  white.  It  is  then  dissolved  in  a  small  quantity  of 
water,  filtered,  and  the  filtrate,  after  the  addition  of  a  few 
drops  of  HC1,  is  brought  to  boiling  and  treated  with  BaCl3 
solution.  What  is  this  white  precipitate?  Write  the  equa- 
tion. 

Phosphorus. — Heat  some  casein  with  the  fusion  mixture 
as  in  the  previous  experiment.  Dissolve  the  fused  mass  in 
10  c.c.  of  water  acidified  with  HN03.  Filter  and  add  5  c.c. 
of  ammonium  molybdate  solution.  Warm  some  minutes  at 
about  80°  C.  What  is  the  yellow  precipitate? 

Iron. — Incinerate  a  small  quantity  of  haemoglobin  in  a 
porcelain  crucible.  The  ash  should  be  red.  Dissolve  out 
the  ash  with  10  c.c.  of  dilute  HC1.  Filter.  Test  this  solu- 
tion for  Fe  with  potassium  sulphocyanide  or  potassium 
ferrocyanide. 

Color  Reactions. 

The  following  tests  are  based  upon  reactions  taking  place 
between  the  reagent  employed  and  certain  more  or  less  well- 
defined  groups  or  atomic  complexes  of  the  protein  molecule. 
These  groups  are  present  in  varying  proportions  in  the  dif- 
ferent proteins  and  in  some  cases  even  certain  complexes 


18  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

may  be  lacking  altogether,  facts  which  explain  the  individual 
differences  in  the  strengths  of  the  reactions  obtained. 

For  the  following  tests  make  use  of  the  egg-white  solution 
as  an  example  of  a  typical  protein: 

(a)  Xanthoproteic  Test. — To  5  c.c.  of  the  protein  solution  (or 
dry  substance)  add  5  c.c.  of  HNO3.  Note  the  white  precipi- 
tate. Boil  until  the  precipitate  is  dissolved  and  the  solution 
becomes  light  yellow.  Cool,  and  add  an  excess  of  NH4OH. 
The  color  changes  to  orange.  Other  substances  may  give  a 
yellow  solution  with  HN03,  but  their  solutions  do  not  respond 
orange  upon  neutralization  with  NH4OH.  What  are  the 
chemical  changes  of  this  reaction  and  the  atomic  complexes 
involved? 

(6)  Millon's  Test. — Add  a  few  c.c.  of  Millon's  reagent  to 
5  c.c.  of  the  protein  solution.  The  precipitate  which  forms 
turns  red  slowly  upon  heating.  This  reaction  is  suitable  for 
solids,  or  liquids  when  the  quantity  of  salts  in  the  solution  is 
not  excessive.  What  atomic  complex  in  the  protein  molecule 
causes  this  reaction?  What  simple  substance  also  responds 
to  this  test? 

(c)  Biuret  Test. — Suited  for  testing  solutions  only. — Place 
5  c.c.  of  the  protein  solution  in  a  test-tube  and  add  an  equal 
volume  of  NaOH.     Then  add,  drop  by  drop,  a  CuS04  solu- 
tion so  dilute  that  the  color  is  hardly  visible.     The  color 
obtained  will  vary  from  blue  violet  to  reddish  violet,  accord- 
ing to  the  nature  of  the  protein  in  solution.     If  too  much 
CuS04  is   added  the   solution   becomes   green.     A  specific 
atomic  complex  in  the  protein  molecule  is  also  accountable 
for  this  reaction.     What  simple  substance  reacts  positively 
to  this  test? 

(d)  Adamkiewicz   Test. — To  3   c.c.   of  a  glyoxylic  acid 
solution  (Hopkins-Cole  reagent)  add  1-2  c.c.  of  the  protein 
solution.     Stratify  this  mixture  upon  5  c.c.  of  cone.  H2S04. 


THE  PROTEINS.  19 

A  red  to  reddish- violet  color  develops  at  the  junction  of  the 
two  liquids.  Of  what  important  complex  does  this  reaction 
show  the  presence? 

Precipitation  Reactions. 

The  precipitation  reactions  may  be  divided  into  three 
general  classes  according  as  the  protein  acts  as  a  base,  as  an 
acid,  or  as  a  neutral  body.  To  the  first  class  belong  those 
reactions  in  which  the  weak  organic  acids  combine  with  the 
protein  as  a  base  and  an  insoluble  salt  results.  The  second 
class  of  reactions  embrace  those  between  neutral  salts  and 
the  protein  in  which  an  albuminate  of  the  base  (metal)  is 
formed.  In  the  third  class  the  protein  does  not  take  part 
in  the  reaction.  Precipitation  occurs  when  the  reagent 
which  is  added  (in  this  case  a  salt  such  as  MgS04,  Na^SO^, 
(NH4)2S04)  has  appropriated  to  itself  sufficient  of  the  solvent 
to  throw  the  protein  out  of  its  solution  (salted  out). 

(a)  Heller's  Test. — If  5  c.c.  of  HN03  is  placed  in  a  test- 
tube  and  a  few  c.c.  of  the  albumin  solution  allowed  to  flow 
gently  down  the  side  of  the  test-tube  and  stratify  itself  on 
top  of  the  HN03,  a  white  ring  of  precipitated  albumin  will 
form  where  the  two  liquids  meet.  HC1  and  H2S04  will  also 
give  the  same  reaction,  but  the  precipitates  are  soluble  in  an 
excess  of  the  reagents. 

(6)  Acetic  acid  and  potassium  ferrocyanide. — Make  5  c.c. 
of  the  protein  solution  acid  with  acetic  acid  and  add,  drop 
by  drop,  a  dilute  solution  of  potassium  ferrocyanide.  Note 
result. 

(c)  Make  25  c.c.  of  the  protein  solution  slightly  acid, 
using  dilute  acetic  acid. 

1.  To  5  c.c.  of  this  solution  add  2  drops  of  tannic  acid 
solution. 


20  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

2.  To  5  c.c.  of  this  solution  add  2  drops  of  picric  acid 
solution. 

3.  To  5  c.c.  of  this  solution  add  3  volumes  of  95  per  cent 
alcohol. 

4.  To  5  c.c.  of  this  solution  add  MgS04   (in  substance) 
to  saturation. 

5.  To  5  c.c.  of  this  solution  add  (NH4)2S04  (in  substance) 
to  saturation. 

Note  results  and  see  if  the  precipitation  is  complete  in  each 
case. 

(d)  Acidify  10  c.c.  of  the  protein  solution  with  dilute  HC1. 

1.  To  5  c.c. of  this  solution  add  2  drops  of  phosphotungstic 
acid. 

2.  To  5  c.c.  of  this  solution   add  2   drops   of  potassio- 
mercuric-iodide. 

(e)  To  successive  portions  of  5  c.c.  of  the  protein  solution 
add  a  few  drops  of  CuS04;   neutral  and  basic  lead  acetate; 
HgCl2;  trichloracetic  acid  (2-5  per  cent  solution);  Fe2Cl6. 

Make  careful  notes  of  the  results  of  the  above  reactions 
and  where  possible  write  equations.  Decide  as  to  which 
class  of  reaction  each  belongs. 

The  use  of  metallic  salts  as  antidotes  in  cases  of  poisoning 
is  based  upon  these  precipitation  reactions. 

SIMPLE   PROTEINS. 

The  group  of  simple  proteins  allows  of  a  further  subdi- 
vision into  the  simple  native  proteins  and  the  albuminoids. 

The  native  proteins,  as  far  as  it  can  be  ascertained,  exist 
in  the  fluids  and  tissues  of  the  body  in  the  same  or  at  least  a 
similar  form  in  which  they  appear  in  the  laboratory.  The 
albuminoids  are  chemically  closely  related  but  are  charac- 
terized by  great  insolubility  in  all  neutral  solvents.  To 


THE  PROTEINS.  21 

-the  former  sub-group  belong  the  albumins,  the  globulins, 
histones,  and  protamines;  to  the  latter  the  keratins, 
elastins,  collagen,  recticulin,  and  the  skeletins. 

ALBUMINS. 

The  albumins  present  the  most  characteristic  type  of  the 
proteins  since  they  give  a  positive  response  to  all  the  typical 
protein  reactions. 

Remember  that  an  albumin  (egg-white)  solution  was 
employed  for  all  the  protein  tests. 

(a)  To  10  c.c.  of  the  albumin  solution  add  (NH4)2SO4  to 
saturation.  Note  result  and  compare  with  experiment  (c)  5, 
p.  20.  Filter  off  the  precipitate  and  test  the  filtrate  with  the 
biuret  test. 

(6)  To  10  c.c.  of  the  albumin  solution  add  MgS04  to  sat- 
uration. Compare  this  result  with  that  of  experiment  (c)  4, 
p.  20.  Now  add  2  drops  of  acetic  acid.  What  is  the  precipi- 
tate? 

(c)  Test  the  albumin  solution  for  lead-blackening  sul- 
phur. 

Coagulation. 

Certain  of  the  proteins  in  the  presence  of  water  and  heat 
undergo  a  change  which  is  confined  to  an  intramolecular 
rearrangement  of  the  atoms  in  the  molecule.  This  trans- 
formation, which  is  termed  coagulation,  brings  about  physical 
differences  in  the  behavior  of  the  proteins.  They  become 
insoluble  in  all  of  the  ordinary  protein  solvents.  Under  simi- 
lar conditions  the  degree  of  temperature  at  which  a  definite 
protein  will  coagulate  is  fairly  constant,  a  fact  which  is 
employed  to  ascertain  not  only  the  character  but  also  the 
purity  of  an  unknown  protein. 


22  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

(d)  Heat  5  c.c.  of  the  albumin  solution  to  boiling  and 
then  add  one  or  two  drops  of  very  dilute  acetic  acid.    The 
albumin  separates  out  in  an  insoluble,  flocky  (coagulated) 
form.    Why  is  the  addition  of  the  acetic  acid  necessary  and 
why  is  it  added  after  boiling?    Try  to  dissolve  the  coagulum 
in  some  of  the  ordinary  protein  solvents.     Make  5  c.c.  of  the 
albumin  solution  faintly  alkaline,  and  heat.     Note  differ- 
ences. 

(e)  The  temperature   of    coagulation  is   determined   as 
follows:' 

Fill  a  test-tube  (one-third  of  its  capacity)  with  the  clear, 
very  slightly  acidulated  solution  of  the  protein.  By  means 
of  a  bored  cork  fasten  into  the  test-tube  a  thermometer  in 
such  a  manner  that  its  bulb  is  entirely  immersed  in  the  solu- 
tion. Suspend  the  test-tube  in  a  large  beaker  of  water  which 
rests  upon  a  wire  gauze.  By  cautious  heating,  the  tempera- 
ture of  the  water  in  the  beaker  may  be  raised  slowly  and  the 
point  on  the  thermometer  noted  at  which  the  first  flocks  of 
coagulated  protein  appear  in  the  solution.  This  point  is 
considered  as  the  coagulation  temperature  of  the  substance 
under  these  conditions,  but  it  varies  with  the  reaction  of 
the  solution  and  the  nature  and  quantity  of  the  salts 
present. 

Another  form  of  coagulation  is  induced  by  the  action  of 
certain  ferments,  such  as  the  fibrin  ferment  of  the  blood,  by 
the  action  of  which  the  soluble  fibrinogen  is  transformed  into 
insoluble  fibrin. 

(/)  Heat  some  dried  albumin  in  a  dry  test-tube  to  about 
100°  C.  After  cooling  the  tube  try  to  dissolve  the  substance 
in  water.  Why  does  not  the  albumin  coagulate  by  the 
heat? 


THE  PROTEINS.  23 

GLOBULINS. 

As  a  class  the  globulins  are  characterized  by  their  insolu- 
bility in  water  and  solubility  in  weak  salt  solutions  (5-10  per 
cent).  A  suitable  solution  of  a  typical  globulin  (edestin)  is 
easily  prepared  by  extracting  finely  ground  hemp-seed  for 
an  hour  with  a  10  per  cent  solution  of  NaCl,  and  finally 
filtering  the  mixture  through  paper.  The  edestin  may  be 
obtained  in  crystalline  form  if  the  extraction  of  the  seed  is 
made  with  a  5  per  cent  solution  of  NaCl  at  55°  C.  and  the 
extract  filtered  through  a  hot-water  funnel.  Upon  cooling 
the  protein  separates  out  of  the  solution  in  well-formed  and 
characteristic  crystals,  hexagonal  in  shape. 

(a)  Try  two  protein  color  reactions  and  three  precipita- 
tion reactions.  Test  the  coagulability  of  the  solution. 

(6)  Pour  some  of  the  solution,  drop  by  drop,  into  a  beaker 
of  water.  A  precipitation  of  the  globulin  occurs  owing  to 
the  decrease  in  the  percentage  of  salt  in  the  solution  by  dilu- 
tion. A  similar  precipitation  may  also  be  accomplished  by 
removing  the  salts  by  dialysis. 

(c)  Start  dialysis  experiment.    A  very  simple  form  of 
dialyzer  can  be  arranged  as  follows: 

Select  a  beaker  and  a  funnel  whose  stem  has  been  removed 
at  the  neck  and  whose  diameter  is  somewhat  greater  than 
that  of  the  beaker.  Allow  the  funnel  to  hang  in  the  beaker 
resting  upon  its  rim.  Cut  and  fold  some  well-moistened 
parchment  paper  in  the  manner  of  a  filter  and  place  it  in  the 
funnel.  Fill  the  beaker  nearly  to  the  top  with  water  and 
pour  into  the  parchment  filter  enough  of  the  solution  to  be 
dialyzed  so  that  it  about  half  fills  it.  The  levels  of  the  inner 
and  outer  fluids  should  correspond. 

In  24  hours  note  carefully  any  changes  which  may  have 
occurred.  What  has  caused  them? 

(d)  Saturate  10  c.c.  of  the  globulin  solution  with  MgS04. 


24  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

Filter  off  the  precipitate  and  test  the  filtrate  for  protein. 
Compare  this  reaction  with  the  similar  one  for  albumin. 

(e)  Cool  10  c.c.  of  the  globulin  solution  to  about  0°  C. 
and  let  C02  gas  bubble  through  it.  Note  result. 

(/)  Take  10  c.c.  of  blood-serum  and  saturate  it  with 
MgS04.  What  is  this  precipitate?  Filter,  and  to  the  fil- 
trate add  two  drops  of  acetic  acid.  What  is  this  second 
precipitate?  Test  both  precipitates  for  protein. 

ALBUMINOIDS. 

The  albuminoids  form  a  class  of  substances  of  hetero- 
geneous nature  closely  allied  to  the  proteins,  but  exhibiting 
also  marked  differences.  They  form  the  chemical  basis  of 
the  skeletal  and  epidermal  structures  and  the  variations  in 
the  properties,  and  characteristics  which  are  observed  in 
them  are  probably  dependent  upon  morphological  changes. 
In  general  they  occur  in  an  insoluble  form  and  are  charac- 
terized by  their  resistance  to  reagents  which  tend  to  dissolve 

them. 

COLLAGEN. 

Collagen  occurs  as  the  chief  constituent  of  connective 
tissue  (e.g.,  cartilage)  and  also  as  the  organic  matrix  of  bone 
(ossein).  It  is  easily  hydrated  by  boiling  with  water  or 
weak  acids  and  is  then  converted  into  gelatin,  which  on 
this  account  may  be  considered  as  the  hydrate  of  collagen. 

GELATIN. 

Gelatin  possesses  the  characteristic  of  forming,  with  hot 
water,  solutions  which  set  in  a  jelly  upon  cooling,  if  the  con- 
centration is  greater  than  1  per  cent. 

Finely  cut  tendons  or  bones  from  which  the  salts  have 


THE  PROTEINS.  25 

been  previously  removed  by  allowing  them  to  remain  in  weak 
HC1  for  several  days,  serve  as  excellent  material  for  the 
preparation  of  gelatin. 

Place  the  substance  in  an  evaporating-dish  half  full  of 
slightly  acidulated  water  and  continue  to  boil  until  the 
material  is  dissolved.  Do  not  allow  the  solution  to  become 
concentrated.  Prolonged  boiling  will  also  cause  the  gelatin 
to  be  converted  into  gelatoses  which  do  not  possess  the 
power  of  gelatinization. 

Make  the  following  tests,  dissolving  the  jelly  in  hot  water 
as  it  is  needed: 

(a)  Biuret;  (6)  Millon's;  (c)  Acetic  acid  and  potassium 
ferrocyanide;  (d)  Tannic  acid;  (e)  HC1  or  H2S04;  (/)  Satu- 
ration with  (NH4)2S04;  (g)  Lead-blackening  sulphur;  (h) 
Adamkiewicz.  Note  and  compare  the  results  carefully  with 
those  obtained  with  proteins. 

KERATINS. 

These  form  the  chief  constituent  of  hair,  nails,  hoofs,  horns, 
feathers,  etc.    Their  main  characteristic  is  insolubility,  and  the 
relatively  large   percentage  of  sulphur  which  they  contain. 
This  is  probably  present  in  a  cystine  nucleus  in  the  molecule, 
and  the  largest  part  is  given  off  in  the  lead-blackening  form. 
Use  horn  shavings  for  the  following  reactions: 
(a)  Lead-blackening  sulphur;    (6)  Millon's;    (c)  Xantho- 
proteic;  (d)  Adamkiewicz;   (e)  Try  its  solubility  in  water, 
dilute  acids,  and  alkalies. 


26    LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

CONJUGATE   PROTEINS. 

These  substances  are  much  more  complex  in  their  chem- 
ical constitution  than  the  simple  proteins.  They  may  be 
subdivided  into — 

1.  The  Glycoproteins — compounds  of  a  protein  component 
and  a  carbohydrate  complex. 

2.  The  Nucleoproteins — compounds  of  a  protein  compo- 
nent and  nucleins  or  nucleic  acids. 

3.  The  Hcemoglobins  and  related  bodies — compounds  of 
a  protein  component  and  a  pigment  which  is  chiefly  respira- 
tory in  character. 

4.  The    Lecithoproteins — compounds    of   a   protein    com- 
ponent with  lecithins. 

5.  The  Phosphoproteins — compounds  of  a  protein  com- 
ponent and  a  phosphorus  containing  substance  other  than 
nucleic  acid  or  lecithins. 

GLYCOPROTEINS. 

In  a  certain  sense  some  of  the  simple  proteins  may  be 
considered  as  glycoproteins,  in  that  they  yield,  upon  decom- 
position, reducing  substances  of  undoubted  carbohydrate 
nature;  in  the  case  of  the  true  glycoproteins,  however,  the 
carbohydrate  nucleus  is  obtained  without  interference  with 
the  integrity  of  the  component  protein  molecule.  The 
mucins,  mucoids,  and  chondroproteins  form  the  chief  classes 
of  the  glycoproteins. 

Of  the  mucins,  that  obtained  from  the  saliva  is  selected 
as  a  typical  example  for  study.  They  do  not  contain  phos- 
phorus, are  acid  in  nature,  and  are  mainly  characterized  by 
forming  viscous  colloidal  solutions.  The  mucins  dissolve  in 
weak  alkali  solutions  and  may  be  reprecipitated  on  the  addi- 
tion of  a  weak  acid.  Upon  boiling  with  a  dilute  acid  they  split 
off  a  carbohydrate  nucleus  which  reduces  Fehling's  solution. 


THE  PROTEINS.  27 

Add  100  c.c.  of  saliva  to  200  c.c.  of  95  per  cent  alcohol. 

Filter  off  the  precipitated  mucin  and  make  the  following 
tests : 

(a)  Try  some  color-reactions  for  proteins. 

(6)  Dissolve  some  of  the  precipitate  in  weak  NaOH  and 
then  add  dilute  acetic  acid,  drop  by  drop.  Note  results. 

(c)  Boil  the  remaining  precipitate  for  10  minutes  in  a 
small  flask  with  50  c.c.  of  5  per  cent  HC1,  cool,  neutralize 
with  NaOH,  and  test  for  a  reducing  body  with  Fehling's 
solution. 

THE  NUCLEOPROTEINS  AND  NUCLEINS. 

As  the  name  indicates,  the  nucleoproteins  originate  in 
and  are  derived  from  the  so-called  mitoplasm  or  chromatin 
of  the  cell  nucleus  and  consequently  exist  in  greater  quanti- 
ties in  the  glandular  organs  than  elsewhere  in  the  body. 
Their  individual  nomenclature  is  founded  upon  their  place 
of  origin;  thus  the  pancreas,  spleen,  and  yeast  nucleopro- 
tein.  By  boiling  with  a  weak  acid  they  are  decomposed 
into  a  protein  component  and  the  nucleins  which,  in  turn, 
upon  further  boiling,  split  up  into  another  protein  part  and 
the  nucleic  acids.  The  nucleoproteins  and  nucleins  only 
differ,  therefore,  in  the  quantity  of  the  protein  component 
which  they  contain,  and  since  the  nucleic  acid  radical  is  the 
carrier  of  all  the  phosphorus  which  is  present  in  the  com- 
pound, it  follows  that  the  nucleins  are  chemically  distin- 
guishable from  the  nucleoproteins  by  their  larger  phosphorus 
content.  The  two  terms  are  sometimes  used  interchangea- 
bly. The  nucleins  are  acidic  in  character,  soluble  in  alkalies, 
but  are  precipitated  by  acids  and  alcohol.  Upon  treatment 
with  caustic  alkalies,  nucleic  acids  result, — substances  which 
contain  phosphorus  in  the  form  of  phosphoric  acid.  The 


28    LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

nucleic  acids  vary  according  to  their  content  of  carbohydrate 
nucleus,  purine  and  pyrimidine  bases. 

Impure  nucleins  may  be  prepared  for  study  by  thorough 
digestion  of  a  nucleoprotein-containing  tissue  with  pepsin- 
hydrochloric  acid  and  extraction  of  the  residue  with  NH4OH. 
The  nuclein  is  then  precipitated  from  the  solution  with 
dilute  HC1. 

(a)  Try  the  solubility  of  the  substance  in  water,  dilute 
alkali  and  alcohol. 

(6)  Try  three  color  protein  tests. 

(c)  Boil    some  with  10  per  cent  H2S04  for  5  minutes, 
cool,  add  an  excess  of  NH4OH,  and  finally  a  few  drops  of 
AgN03  solution.     Of  what  is  the  precipitate  indicative? 

(d)  Treat  a  little  of  the  substance  in  a  crucible  with  some 
fusion  mixture.     Test  the  fused  mass  for  phosphorus. 

DERIVED  PROTEINS. 

These  substances  may  be  subdivided  into  primary  and 
secondary  derivatives  according  as  the  hydrolytic  change  is 
slight  or  more  deep  seated. 

1.  PRIMARY  PROTEIN  DERIVATIVES. 
(A)  Metaproteins. — Products  of  the  action  of  acids  and 
alkalies  whereby  the  protein  molecule  is  so  far  altered  as  to 
form  products  soluble  in  very  weak  acids  and  alkalies  but 
insoluble  in  neutral  fluids. 

ACID  AND  ALKALI  ALBUMINATES. 

The  albuminates  are  modified  proteins  derived  from 
either  albumins  or  globulins  by  the  action  of  acids  and  alkalies 
with  the  aid  of  heat.  They  are  non-coagulable  by  heat 
except  when  held  in  suspension  in  the  solutions  from  which 
they  have  been  precipitated  by  neutralization  respectively 


THE  PROTEINS.  29 

with  acids  or  alkalies.  In  the  formation  of  alkali  albuminate 
neutral  sulphur  is  split  off  from  the  protein  molecule. 

To  25  c.c.  of  the  albumin  solution  add  5  drops  of  very 
dilute  HC1  and  warm  for  15  minutes  at  40°  C.  Cool. 

(a)  Exactly  neutralize  one-half  of  this  solution  with  very 
dilute  NaOH.  What  is  this  precipitate?  Shake  up  the  pre- 
cipitate and  divide  the  solution  into  two  parts.  To  the  first 
add  a  drop  of  NaOH  and  heat.  Heat  the  other  part  and 
then  add  a  drop  of  NaOH.  Explain  the  differences  in  results. 

(6)  Try  three  characteristic  protein  tests  on  the  remain- 
ing half  of  the  solution. 

To  25  c.c.  of  the  albumin  solution  add  10  drops  of  NaOH 
and  warm  for  15  minutes  at  40°  C.  Cool. 

(c)  Try  the  effect  of  heating  some  of  this  solution. 

(d)  Neutralize    the    solution  and  repeat  the  procedure 
employed  under  (a),  using  HC1  instead  of  NaOH. 

(e)  Try  three  characteristic  protein  tests. 
Explain  the  results. 

(B)  Coagulated  Proteins. — Insoluble  products  which  result 
from  (1)  action  of  heat  on  their  solutions,  (2)  action  of  alco- 
hols on  the  protein.  Heat  coagulation  has  been  considered 
on  p.  21. 

2.  SECONDARY  PROTEIN  DERIVATIVES. 
Products  of  a  more  pronounced  hydrolytic  cleavage  of 
the  protein  molecule. 

(A)  Proteases,  see  p.  52. 

(B)  Peptones,  see  p.  53. 

(C)  Peptides.— These  are  definitely  characterized   com- 
binations of  two  or  more  amino-acids,  the  carboxyl  group  of 
one  being  united  with  the  arnino  group  of  the  other  with  the 
elimination  of  a  molecule  of  water.     Some  of  the  more  com- 
plex polypeptides  give  the  biuret  reaction. 


MUSCULAR  TISSUE. 

The  chief  chemical  constituents  of  muscular  tissue  may 
be  divided  as  follows: 

The  proteins  of  which  the  two  most  important  are  myogen, 
an  albumin  (not  typical;,  and  myosin,  a  globulin. 

The  nitrogenous  extractives,  comprising  creatine,  purine 
bases,  uric  acid,  carnine,  and  urea. 

The  non-nitrogenous  extractives,  made  up  of  glycogen, 
dextrose,  lactic  acid,  inosit,  fats,  and  inorganic  salts. 

Reaction. — Test  the  reaction  of  living  and  dead  muscle  to 
litmus  and  Congo-red  paper.  Explain  the  differences  in  the 
results  obtained. 


PROTEINS. 

MYOGEN. 

Place  25  grms.  of  hashed  fresh  muscle  in  a  beaker  with  75 
c.c.  of  water  and  allow  it  to  stand,  with  frequent  stirring,  for 
one  hour.  Strain  off  the  muscle  through  some  cheese-cloth 
(keep  the  residue),  and  filter  the  solution. 

Use  this  solution  for  the  following  tests: 

(a)  What  is  the  reaction  of  the  solution? 

(6)  Test  the  coagulability  of  the  protein  in  the  solution. 

(c)  Perform  three  color  protein  tests. 

(d)  Is  the  protein  precipitated  by  MgSO4  or  (NH4)2S04? 
Why  is  myogen  not  a  typical  albumin  or  globulin? 

30 


MUSCULAR  TISSUE.  31 

MYOSIN. 

Digest  the  above  meat  residue  with  100  c.c.  of  15  per  cent 
NH4C1  solution  for  24  hours  in  a  covered  beaker.  Then  filter 
and  use  the  filtrate  for  the  following  reactions: 

(a)  Pour  a  little  of  the  solution,  drop  by  drop,  into  a 
beaker  filled  with  water.  Compare  this  reaction  with  a  simi- 
lar one  under  Globulins. 

(6)  Heat  a  few  c.c.  of  the  filtrate.  Filter  and  test  the  fil- 
trate for  Ca.  Is  myosin  a  coagulable  protein  ? 

(c)  Saturate  10  c.c.  of  the  filtrate  with  MgS04.     Filter  and 
test  the  filtrate  for  protein. 

(d)  Try  Adamkiewicz,  Biuret,  and  Xanthoproteic  tests. 

NITROGENOUS  EXTRACTIVES. 

These  substances,  together  with  the  non-nitrogenous 
extractives,  form  the  chief  constituents  of  a  hot-water  extract 
of  muscular  tissue,  such  as  is  known  commercially  as  Liebig's 
Extract.  The  most  important  of  the  nitrogenous  extract- 
ives are  creatine,  xanthlne,  hypoxanthine,  guanine,  uric 
acid,  and  urea.  The  latter  two  are  only  present  in  traces. 
The  purine  bases  represent  in  all  probability  an  intermediate 
stage  in  the  nuclear  metabolism  of  the  muscle-cell  between 
the  nucleins  on  the  one  hand  and  uric  acid  or  urea  on  the 
other. 

/NH2 

CREATINE,  HN  =  C<         /CH3 

N\      //° 

XCH2-C< 

XOH 

Extract  500  grms.  chopped  beef  with  500  c.c.  water  for 
half  an  hour  over  the  water-bath  at  50°  C.  Strain  as  dry  as 
possible  through  muslin  and  make  a  second  extraction  in  the 


32  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

same  way,  with  an  equal  amount  of  water  (save  residue). 
Unite  the  two  extracts,  and  after  concentration  to  about  200 
c.c.  acidify  the  solution  with  2  or  3  drops  of  acetic  acid. 
Remove  the  coagulated  proteids  by  filtration,  and  to  the  fil- 
trate *  add  basic  lead  acetate,  carefully  avoiding  any  excess; 
the  precipitate  consists  of  phosphates,  chlorides,  sulphates,  etc. 
Allow  this  to  settle  and  then  filter.  Warm  the  filtrate  and 
pass  H2S  through  it  to  remove  the  excess  of  lead.  Filter  hot. 
The  filtrate,  which  should  be  water-clear,  is  then  concen- 
trated on  a  water-bath  to  a  thin  syrup.  Upon  standing  sev- 
eral days  in  a  cool  place  crystals  of  creatine  will  deposit.  Filter 
off  the  crystals,  and  wash  them  with  88  per  cent  alcohol. 
(Keep  the  filtrate  for  the  separation  of  the  purine  bases.) 

Place  some  of  the  crystals  in  a  small  flask  with  10  c.c. 
dilute  H2SO4,  and  heat  for  half  an  hour  on  the  water-bath, 
keeping  the  volume  constant.  While  still  warm  add  BaC03, 
in  substance,  to  neutralization.  Filter  and  evaporate  the 
filtrate  to  10  c.c.  The  creatine  has  been  changed  to  creatinine. 
Write  the  equation.  Perform  the  following  tests  with  the 
solution: 

(a)  Place  2  drops  of  the  solution  upon  a  watch-glass  and 
add  to  it  a  few  drops  of  an  alcoholic  solution  of  ZnCl2;  allow 
it  to  stand  for  several  days  and  then  examine  the  crystals 
under  the  microscope. 

(6)  Weyl's  Reaction. — To  2  c.c.  of  the  creatinine  solution 
add  three  drops  of  a  freshly  prepared  dilute  solution  of 
sodium  nitroprusside.  Then  add,  drop  by  drop,  dilute 
NaOH.  A  ruby-red  color  is  produced  which  quickly  changes 
to  yellow.  If  the  solution  is  now  acidified  with  acetic  acid 

*  This  filtrate  corresponds  to  Liebig's  extract,  and  if  the  latter  is  used 
for  study  instead  of  chopped  beef,  the  procedure  of  separation  is  taken 
up  at  this  point. 


MUSCULAR   TISSUE.  33 

and  heated,  a  green  color  is  obtained,  and  upon  continued 
boiling  a  precipitate  of  Prussian  blue  settles  out. 

(c)  Jaffe's  Reaction.  —  Treat  some  of  the  solution  with  a 
dilute  solution  of  picric  acid,  and  make  it  faintly  alkaline 
with  NaOH.  The  solution  immediately  becomes  a  deep  red 
Acetone  responds  to  this  test,  but  gives  merely  a  yellowish- 
red  color. 

PURINE  BASES. 

(6) 


(1) 

(2)  HC    (5)C—  NI 


I          I      (7) 

C— 


H  (8) 

(3)     N  -  C  - 

(4)       (9) 

This  represents  structurally  the  compound,  purine,  the 
nucleus  from  which  all  the  purine  bases  may  be  derived.    The 
structure  of  the  bases  is  easily  obtained  by  substitution  in 
the  nucleus  at  the  position  indicated  by  number. 
Hypoxanthine  =  6-Oxypurine. 
Guanine  =  2-Amino-6-oxypurine. 
Xanthine  =  2,6-Dioxypurine. 
Adenine  =  6-Aminopurine. 

The  purine  bases  in  greater  part  exist  as  constituent  groups 
in  the  more  complex  nucleic  acid  molecule.  In  some  tissues, 
however,  they  have  been  found  in  the  free  state.  They  form 
crystalline  salts  with  the  mineral  acids  and  are  all  precipitated 
from  acid  solutions  by  means  of  phosphotungstic  acid. 
Upon  the  addition  of  ammoniacal  silver  nitrate  they  sepa- 
rate from  their  solutions  as  silver  combinations.  Copper 
acetate  combines  with  them  to  form  insoluble  compounds. 

The  separation  of  the  bases  from  the  creatine  filtrate  (see 
p.  31)  takes  place  as  follows: 


34    LABORATORY  WORK  IX  PHYSIOLOGICAL  CHEMISTRY. 

After  removal  of  the  alcohol,  the  filtrate  is  made  ammo- 
niacal  with  NH4OH  and  ammoniacal  AgN03  solution  added 
until  no  further  precipitation  occurs.  This  precipitate  is 
composed  of  the  double  silver  salts  of  all  the  purine  bases. 
These  silver  combinations,  after  their  removal  from  the 
solution  by  filtration,  are  dissolved  in  boiling-hot  HX03 
(sp.  gr.  1.1)  and  the  mixture  filtered  hot. 

In  this  reaction  the  silver  nitrate  compounds  of  the  bases 
are  formed,  all  of  which  are  soluble  in  the  hot  nitric  acid  solu- 
tion. Upon  cooling,  the  guanine,  adenine,  and  hypoxan- 
thine  combinations  crystallize  out,  but  the  xanthine  remains 
in  the  solution,  from  which,  after  filtration  and  treatment 
with  NH4OH  in  excess,  it  is  thrown  down  as  a  reddish 
precipitate  (xanthine  silver  oxide).  This  precipitate,  sus- 
pended in  its  solution,  is  next  treated  with  hydrogen  sulphide 
and  the  mixture  warmed  and  filtered.  The  xanthine  will 
crystallize  out  of  the  filtrate  upon  concentration. 

The  precipitate  of  the  three  other  bases  is  suspended  in 
water  and  treated  with  hydrogen  sulphide,  warmed  and 
filtered  hot  in  the  same  manner  as  the  xanthine.  After  con- 
centration the  solution  is  saturated  with  NB^OH  and  digested 
on  the  water-bath.  The  guanine  remains  insoluble,  while 
the  other  two  bases  pass  into  solution.  Filter  the  mixture 
hot.  From  this  filtrate  when  freed  from  the  ammonia  and 
allowed  to  cool,  the  adenine  separates,  leaving  the  hypoxan- 
thine  in  the  solution. 

(a)  Try  some  of  the  crystals  with  murexide  test. 

(6)  Examine  the  various  precipitates  for  crystals.  Make 
sketches  of  them. 

(c)  Warm  some  of  the  xanthine  crystals  with  bromine- 
water  and  evaporate  the  solution  to  dryness  on  the  water- 
bath.  Allow  ammonia  vapors  to  come  in  contact  with  the 
residue;  this  becomes  red. 


MUSCULAR   TISSUE.  35 

NON-NITROGENOUS   EXTRACTIVES. 
GLYCOGEN  (CftH1005)n. 

This  polysaccharide,  sometimes  called  animal  starch  on 
account  of  its  similarity  in  function  to  plant  starch,  exists 
wherever  living  protoplasm  is  present  and  is  therefore 
classed  as  one  of  the  primary  constituents  of  the  cell.  It  is 
especially  abundant  in  the  liver,  where  it  apparently  repre- 
sents the  carbohydrate  surplus  of  the  organism  and  in  the 
muscle,  where  it  serves  as  a  ready  source  of  energy.  Glyco- 
gen  forms  a  tasteless  white  powder,  insoluble  in  alcohol  and 
ether,  but  forming  with  water  an  opalescent  solution  which 
is  dextrorotatory.  It  does  not  reduce  metallic  oxides  in 
alkaline  solution.  By  the  action  of  hydrolytic  agents  or 
enzymes  it  is  converted  into  maltose  and  dextrose  similarly 
to  starch. 

The  common  scallop  serves  as  the  most  convenient  and 
prolific  source  of  glycogen  from  muscular  tissue.  By  the 
simple  extraction  of  the  tissues  with  boiling  water,  slightly 
acidulated,  a  solution  is  obtained  for  the  following  reactions: 

(a)  Notice  the  color  of  the  solution.  Add  2  drops  of  the 
iodine  solution  to  5  c.c.  of  the  glycogen  solution.  Warm 
gently  and  allow  to  cool.  Note  changes. 

(6)  Test  5  c.c.  of  the  solution  with  Fehling's  solution. 

(c)  To  5  c.c.  of  the  solution  add  three  volumes  of  95  per 
cent  alcohol. 

(d)  To  5  c.c.  of  the  solution  add  5  drops  of  concentrated 
HC1  and  boil  for  some  minutes.    Neutralize  cold  with  NaOH 
and  test  with  Fehling's  solution.     What  is  this  reducing 
substance?    How  could  you  prove  it? 

(e)  To  5  c.c.  of  the  solution  add  a  few  drops  of  filtered 
saliva,  and  warm  at  40°  C.  for  a  few  minutes.     Notice  changes 
in  the  solution.    Determine  the  character  of  the  final  reducing 
body  formed. 


BONE. 

Bone  consists  of  an  organic  matrix,  ossein,  which  is  identi- 
cal with  collagen  obtained  from  the  white  fibrous  connective 
tissue.  This  matrix  is  impregnated  with  insoluble  inorganic 
salts,  which  serve  to  give  strength  and  stability  to  the  tissue. 
Ossein  when  hydrated  with  weak  acids  is  converted  into 
gelatin. 

MINERAL  CONSTITUENTS. 

Incinerate  10  grms.  of  bone  under  the  hood.  Extract  the 
grayish  residue  with  25  c.c.  of  hot  dilute  HN03,  filter,  and 
use  the  filtrate  for  the  following  tests: 

(a)  Phosphoric  Acid. — To  10  c.c.  of  the  solution  add  10  c.c. 
of  the  molybdic  solution  and  warm  in  the  water-bath  at  75° 
C.  until  a  canary-colored  precipitate  settles  out.  What  is 
this?  Filter  it  off  on  a  small  filter,  wash  once  with  very 
dilute  HN03,  and  then  dissolve  the  precipitate  upon  the 
filter  by  adding  dilute  NH4OH,'  drop  by  drop.  To  the  fil- 
trate add  a  few  c.c.  of  magnesium  mixture.  What  is  this 
precipitate?  Write  the  equations  for  all  the  reactions  in 
this  procedure. 

(6)  Chlorides. — To  a  few  c.c.  of  the  solution  add  a  few 
drops  of  AgN03.  What  results? 

(c)  Calcium. — Make  10  c.c.  of  the  solution  alkaline  with 
NH4OH.  What  is  the  precipitate?  Filter,  and  to  the  filtrate 
add  ammonium  oxalate.  The  white  precipitate  of  calcium 

36 


BONE.  37 

oxalate  is  proof  of  the  presence  of  calcium  combined  other- 
wise than  with  phosphoric  acid. 

(d)  Magnesium. — To  15  c.c.  of  the  solution  add  a  slight 
excess  of  NH4OH.     Then  make  the  mixture  acid  with  acetic 
acid;  this  should  dissolve  the  greater  part  of  the  precipitate. 
The  slight  insoluble  substance  is  to  be  filtered  off,  dissolved 
in  HC1  and  tested  for  iron  by  means  of  potassium  ferro- 
cyanide.    The  acetic  acid  filtrate  is  treated  with  a  few  c.c. 
of  ammonium  oxalate  and  the  resultant  precipitate  filtered 
off.    This  new  filtrate  is  made  alkaline  and  a  few  c.c.  of  a 
disodium  phosphate  solution  added.    Why  does  a  precipitate 
at  this  point  prove  the  presence  of  magnesium?    Write  the 
equations. 

(e)  Carbonates. — Did  you  notice  an  effervescence  when 
you  added  HN03  after  incineration?    What  was  the  cause? 


NERVOUS  TISSUES. 

Nervous  tissue  presents  for  chemical  study  the  following 
classes  of  substances: 

1.  Proteins — two  ordinary  cell  globulins    coagulating   at 
45°  C.  and  75°  C.,  and  a  nucleoprotein. 

2.  Lipoids — bodies  of  a  fatty  nature,  including  the  leci- 
thins, cerebrosides,  and  protagon.     It  is  somewhat  question- 
able whether  the  latter  substance  is  a  unit  or  a  mixture  of 
lecithins,  cer^brin,  and  proteins. 

3.  Cholesttrols  and  fatty  acids. 

4.  Neurokeratin — a  substance  apparently  related  to  ker- 
atin. 

5.  Extractives — the  same  as  those  of  muscular  tissue. 


LIPOIDS. 

The  lipoids,  together  with  the  cholesterols,  are  usually 
classed  as  the  myelin  bodies  from  the  fact  that  they  exist  as 
a  mixture  in  the  so-called  myelin  substance  of  the  medulla. 
They  contain  fatty  acid  radicals  in  their  molecule,  and  on 
this  account  are  closely  related  to  the  fats  and  sometimes 
are  classified  under  them.-  For  the  sake  of  convenience  in 
isolation  for  study  they  will  be  considered  here,  although  it 
must  not  be  forgotten  that  the  lecithins  are  widely  distributed 
in  all  forms  of  living  tissues  and  belong  to  the  primary  con- 

38 


NERVOUS  TISSUES.  39 

stituents  of  cells  in  general.  The  cerebrosides  are  found  in 
plant  as  well  as  animal  tissues.  So  little  that  is  positive 
is  known  about ' '  protagon  "  that  no  notice  need  be  taken  of  it. 

LECITHINS. 


CH-O-R' 


CH2-0- 


PO— 0-C2H4\ 

(CH3)=N 
OH  OH/ 

R  and  R'=the  same  or  different  fatty  acid  radicals. 

The  structure  of  the  lecithin  molecule  is  proved  by  the 
•decomposition  products  which  are  obtained  from  it,  viz., 
chohne,  glycerophosphoric  acid,  and  fatty  acids.  From  the 
fact  that  various  fatty  acid  radicals  enter  into  the  composi- 
tion of  the  molecule  and  that  the  nitrogenous  complex  may 
also  differ,  it  follows  that  the  existence  of  many  lecithins  is 
possible.  As  a  class  they  are  soluble  in  ether  and  alcohol 
and  from  such  solutions  may  be  precipitated  by  cooling  to 
0°  C.  or  by  the  addition  of  acetone.  The  lecithins  combine 
with  acids  and  bases  as  well  as  with  proteins  and  other  bodies 
to  form  more  or  less  loose  combinations,  such  as  the  lecitho- 
proteins.  With  water  they  swell  up,  giving  off  long  fila- 
ments (myelin  forms),  and  finally  pass  into  an  emulsion. 

Various  lecithins  can  be  separated  from  the  yolk  of  the 
egg,  from  yeast,  or  from  brain-tissue.  The  procedure  for  the 
isolation  of  the  brain-lecithins  is  as  follows:  The  finely 
divided  brain-tissue,  which  may  or  may  not  have  been  dehy- 
drated by  boiling  with  acetone  for  some  hours  previously,  is 


40  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

allowed  to  stand  for  three  days  in  cold  ether.     The  solution 
is  then  filtered,  and  to  the  filtrate,  which  contains  the  lecithins 
and   cholesterol,  acetone  is  added.     The  precipitated  leci- 
thins may  be  removed  by  filtration  and  the  ether-acetone 
filtrate  evaporated  to  dryness.     (Keep  this  cholesterol.) 
Try  the  following  reactions  with  the  lecithin: 
(a)  Place  a  particle  of  the  substance  on  a  glass  slide  and 
add  a  drop  or  two  of  water.     Examine  this  under  the  micro- 
scope. 

(6)  Shake  some  of  the  substance  in  water  and  examine 
microscopically. 

(c)  To  some  of  the  substance  add  a  drop  of  osmic  acid. 
What  is  the  effect? 

(d)  Test  the  substance  for  phosphorus  by  fusion. 

(e)  Test  for  the  glycerol  radical. 

CEREBROSIDES. 

This  class  of  non-phosphorized  nitrogenous  substances, 
whose  exact  composition  is  not  known,  is  characterized  by  a 
glucoside  constitution  yielding  upon  decomposition  a  sugar 
which  is  identical  with  galactose.  Fatty  acids  in  relatively 
large  quantities  can  also  be  isolated  from  the  cleavage  prod- 
ucts. 

CEREBRIN. 

This  is  the  most  important  substance  in  this  group. 
Whether  it  exists  uncombined  in  the  tissue  is  still  an  un- 
decided question.  It  is  insoluble  in  water,  dilute  alkalies, 
or  baryta-water.  It  dissolves  in  boiling  alcohol,  from  which 
solution  it  separates  as  a  flaky  precipitate  upon  cooling. 
This  precipitate  is  made  up  of  microscopic  globules. 


NERVOUS   TISSUES.  41 

Cerebrin  is  usually  prepared  according  to  the  following 
procedure:  A  small  amount  of  the  finely  divided  brain-tissue 
is  boiled  for  one-half  hour  in  a  casserole  with  twice  its  weight 
of  baryta-water.  The  solution  is  filtered  off  and  the  residue 
extracted  with  boiling  95  per  cent  alcohol.  From  this 
extract,  if  filtered  while  still  hot,  the  cerebrin  will  separate 
in  small  crystals.  The  cholesterol  should  stay  in  the  alco- 
holic solution,  but  if  any  becomes  insoluble  with  the  cere- 
brin it  is  easily  removed  by  washing  the  precipitate  with 
ether  in  the  filter. 

Test  the  substance  as  follows: 

(a)  Try  to  detect  the  presence  of  nitrogen  and  phos- 
phorus. 

(6)  Boil  some  of  the  substance  for  one  hour  with  5  per 
cent  H2S04.  After  cooling  the  mixture,  neutralize  it  and 
test  for  a  reducing  action  on  Fehling's  solution. 

(c)  Warm  some  of  the  substance  on  a  platinum  foil. 
Notice  the  odor.  To  what  is  it  similar? 


THE  CHOLESTEROLS. 

These  bodies  belong  to  the  class  of  primary  cell  con- 
stituents. They  are  studied  here  on  account  of  the  existence 
of  suitable  material  at  hand.  Cholesterol  usually  exists 
free,  as  in  the  case  of  the  brain-tissue  and  gall-stones,  but  it 
is -also  found  in  combination  with  fatty  acids,  as  esters  in 
the  blood-plasma.  The  best  known  cholesterol  is  a  mon- 
atomic  alcohol  with  C27H45OH  as  the  empirical  formula.  At 
one  time  it  was  termed  a  "  non-saponifiable "  fat.  It  has 
the  general  solubilities  of  the  fats  and  crystallizes  readily 
from  an  alcohol  or  ether  solution  in  the  form  of  superimposed 


42  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

rhombic  plates.  The  bile-acids  also  have  a  solvent  action 
on  cholesterol. 

Make  use  of  the  cholesterol  prepared  in  the  course  of  the 
separation  of  lecithin  (p.  39). 

(a)  Try  the  solubility  of  the  substance  in  water. 

(6)  Salkowski's  Test. — Dissolve  a  small  quantity  of  the 
cholesterol  in  a  few  c.c.  of  chloroform  and  add  an  equal 
volume  of  concentrated  H2S04.  The  acid  solution  takes  on 
a  greenish  fluorescence  and  the  chloroform  becomes  red. 

(c)  Liebermann's  Test. — Dissolve  a  crystal  of  cholesterol 
in  about  2  c.c.  of  chloroform  and  add  first  10  drops  of  acetic 
anhydride  and  then  concentrated  H2S04,  drop  by  drop.  The 
mixture  will  become  red,  then  blue,  and  finally  green. 


SALIVARY  DIGESTION. 

As  usually  obtained  for  study,  the  saliva  forms  a  mixture 
of  the  secretion  of  the  submaxillary,  sublingual,  parotid, 
and  the  mucous  glands  of  the  mouth.  It  presents  a  viscid, 
slightly  opalescent  appearance,  possessing  either  alkaline 
or  acid  reaction  according  to  the  indicator  employed,  and 
containing  about  0.5-1  per  cent  solids.  Besides  the  proteins 
and  the  potassium  sulphocyanide,  its  chief  and  essential 
organic  constituent  is  the  enzyme,  ptyalin,  which  gives  to 
the  secretion  its  importance  in  the  digestion  of  carbohydrates. 
A  physical  function  of  the  saliva  consists  in  rendering  the 
food  moist,  which  action,  in  conjunction  with  the  presence  of 
the  mucin,  allows  the  food-bolus  to  be  more  readily  swal- 
lowed. 

Chemical  Characteristics. 

Collect  about  50  c.c.  of  filtered  saliva. 

(a)  Test  its  reaction  with  litmus  paper  and  phenol- 
phthalein.  What  causes  this  apparent  discrepancy? 

(6)  To  a  few  c.c.  of  saliva  add  acetic  acid,  drop  by  drop, 
until  a  precipitate  forms.  (See  under  Mucin.)  Filter  off 
the  mucin  and  test  the  filtrate  for  protein.  What  is  the 
result? 

(c)  Allow  a  drop  of  saliva  to  fall  in  the  centre  of  a  piece 
of  filter-paper.  Then  add  a  drop  of  ferric  chloride  to  the 
paper  where  it  is  moist.  Note  color.  Now  add  a  drop  of 

43 


44  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

mercuric  chloride.     What  is  the  result?    For  what  substance 
are  these  tests?  > 

PTYALIN. 

This  substance  belongs  to  the  class  of  amylolytic  enzymes 
in  that  its  function  consists  in  the  conversion  of  starch  and 
glycogen  into  maltose.  Its  action  is  maximum  at  46°  C. 
and  is  destroyed  at  65-70°  C.  Alkalies  and  free  acids  tend 
to  retard  its  activity.  Apparently  it  acts  best  in  a  neutral 
solution  and  in  the  presence  of  proteins  with  which  the 
acids  or  alkalies  may  combine;  in  this  way  a  greater  degree 
of  acidity  or  of  alkalinity  may  be  borne  without  injury 
than  in  a  pure  solvent.  This  is  important  to  -remember 
when  considering  the  possibility  of  the  continuance  of  saliva 
digestion  in  the  stomach. 

AMYLOLYSIS. 

The  amylolytic  action  of  ptyalin  on  starch  is  a  hydrolytic 
change  in  which  successive  products  are  evolved  until  the 
final  stage  .of  maltose  or  isomaltose  is  reached.  The  end 
product  of  a  similar  cleavage  by  means  of  weak  acids  is 
dextrose.  Thus  from  starch  is  formed  successively  soluble 
starch  or  amygdulin,  then  erythrodextrin,  the  achroodextrins, 
maltodextrin  and  maltose,  and,  in  case  of  acids,  dextrose. 
There  are  indications  that  at  each  step  of  the  cleavage  small 
quantities  of  maltose  are  produced.  These  facts  can  be 
shown  by  the  following  experiment: 

(a)  Prepare  some  starch  paste  and  allow  it  to  cool  to 
40°  C.  Dilute  the  saliva  five  times  and  regulate  the  water- 
bath  for  40°  C.  Take  15  c.c  of  the  paste;  add  to  it  1  c.c.  of 
the  diluted  saliva  and  place  the  test-tube  in  the  water-bath. 
Watch  the  mixture  until  the  turbidity  disappears  and  the 
solution  becomes  transparent.  Then  pour  out  into  a  clean 


SALIVARY  DIGESTION.  45 

test-tube  a  few  c.c.  of  the  solution  and  add  a  drop  of  iodine 
solution.  What  color  is  obtained?  Replace  the  mixture 
in  the  bath  and  as  the  amylolysis  proceeds  remove  successive 
portions  (2  c.c.)  and  test  with  the  iodine  solution.  What 
successive  variations  in  color  do  you  obtain?  Finally  the 
iodine  fails  to  give  a  color  to  the  solution.  This  is  called  the 
achromic  point;  why?  Test  the  remainder  of  the  solution 
with  Fehling's  solution.  What  is  the  character  of  the  sugar 
present?  What  is  the  cause  of  these  changes  indicated  by 
the  iodine  reaction?  Make  a  scheme  of  the  successive 
hydrolytic  products  formed  in  the  digestion. 

Influence  of  Conditions  upon  the  Activity  of  Ptyalin. 

(6)  Make  up  the  following  mixtures  in  separate  test-tubes 
and  keep  them  in  the  water-bath  at  40°  C.,  using  in  each  case 
5  c.c.  of  the  diluted  saliva  and  10  c.c.  of  the  starch  paste. 

(1)  Starch  paste  +  saliva. 

(2)  Starch  paste  +  saliva  first  boiled  and  then  cooled. 

(3)  Starch  paste  +  saliva  exactly  neutralized  with  0.2% 

HC1. 

(4)  Starch  paste  +  saliva  made  acid  with  0.2%  HC1. 

(5)  Starch    paste  +  saliva    made    alkaline    with    0.5% 

Na,C03. 

In  all  these  test-tubes  note  carefully  the  changes  which 
are  taking  place  from  time  to  time,  testing  the  rapidity  of 
digestion  by  means  of  the  iodine  reaction.  Determine  the 
varying  lengths  of  time  necessary  for  the  achromic  point  to 
be  reached. 

After  some  time  it  will  be  noticed  that  in  experiments  (4) 
and  (5)  the  starch  paste  has  not  changed;  change  the  reac-' 
tion  in  each  to  that  corresponding  to  the  normal  saliva  and 
again  allow  them  to  digest.  What  result? 

Make  deductions  from  these  test-tube  experiments. 


GASTRIC  DIGESTION. 

Pure  mixed  gastric  juice  such  as  is  obtained  from  a  gastric 
fistula  forms  a  clear,  colorless,  and  odorless  solution  with 
an  acid  reaction  and  low  specific  gravity  (1.004).  The 
total  solids  amount  to  only  2  per  cent,  of  which  the  following 
are  the  chief. 

Inorganic:  hydrochloric  acid,  the  chlorides  of  sodium, 
potassium,  and  calcium,  with  traces  of  the  phosphates  of 
magnesium  and  iron. 

Organic:  pepsin,  rennin  or  chymosin,  mucin,  traces  of 
other  proteins,  and  sometimes  lactic  acid. 

HYDROCHLORIC  ACID. 

In  all  probability  the  most  important  function  of  the 
hydrochloric  acid  is  that  of  a  germicide  preventing  putrefac- 
tion and  the  formation  of  obnoxious  gases.  Secondarily,  it 
gives  to  the  gastric  contents  a  more  favorable  condition  for 
the  action  of  the  pepsin,  although  the  establishment  of  an 
acid  reaction  is  not  absolutely  necessary  for  peptic  digestion. 
The  hydrochloric  acid  also  seems  to  possess  some  power  of 
inverting  cane-sugar. 

The  following  experiments  are  intended  to  simulate  possi- 
ble gastric  mixtures  which  may  be  encountered  in  testing 
stomach  contents  for  HC1.  The  acid  may  appear  alone  or 
together  with  lactic  acid  and  digestive  products.  The  relia- 
bility and  sensitiveness  of  the  various  indicators  employed 
under  the  different  conditions  will  also  be  shown. 

46 


GASTRIC  DIGESTION.  47 

Test  each  of  the  following  solutions  with  all  the  indicators 
mentioned  below,  and  tabulate  the  results  whether  positive  or 
negative. 

(a)  0.3  per  cent  HC1;  (6)  0.05  per  cent  HC1;  (c)  0.8  per 
cent  lactic  acid;  (d)  a  mixture  containing  equal  volumes  of 
"a"  and  "c";  (e)  a  mixture  containing  equal  volumes  of  "b" 
and  a  2  per  cent  albumose  solution.  Before  using,  warm  the 
last  mixture  for  a  few  minutes  at  40°  C. 


Indicators. 

An  indicator  is  a  substance  which  possesses  a  different 
color  when  dissolved  in  an  alkaline  solution  from  what  it 
does  in  an  acid  one.  They  are  slightly  dissociable  acids  or 
bases,  and  the  change  in  color  is  due  to  the  appearance  or 
disappearance  of  colored  ions. 

1.  Dimethylaminoazobenzene,  N(CH3)2-C6H4-N=N-C6H5. 
Add  one  or  two  drops  directly  to  the  solution  to  be  tested. 
Free  mineral  acid  is  indicated  by  a  carmine-red  color. 

2.  TropceolinOO,   NH(C6H5)  -  C6H4  -  N  =  N  -  C6H4  - 
S03Na.     Add  one  or  two  drops  directly  to  the  solution  to 
be  tested.     Free  acid  is  indicated  by  a  red  or  reddish- violet 
color.    The  reaction  becomes  more  delicate  when  performed 
in  a  similar  manner  to  that  suggested  for  Giinzburg's  reagent. 

3.  Congo-red. — Use  Congo-red  paper,  prepared  by  dipping 
filter-paper  into  the  alkaline  indicator  solution  and  drying. 
Free  acid  is  indicated  by  the  blue  color. 

4.  Gunzburg's  Reagent. — Evaporate  2  drops  of  the  solution 
to  be  tested  with  1  drop  of  the  indicator,  in  a  porcelain  dish, 
carefully,  over  a  water-bath.     Upon  dryness  the  presence  of 
free  HC1  is  indicated  by  the  development  of  a  rose-red  color. 

5.  Boas'  Reagent. — Mix  3  drops  of  the  solution  to  be  tested 
with  the  same  amount  of  indicator  and  evaporate  cautiously 


48  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

in  a  porcelain  dish.    Free  acid  is  shown  by  a  rose  or  vermil- 
ion color. 

/XX  /(OH), 

6.  Alizarin,     C6H4<         >C6H< 

XXX          \S03Na 

Add  2  or  3  drops  of  the  indicator  directly  to  the  solution. 

The  color  will  be  yellow  in  acid  and  acid  salt  solutions,  but 
red  to  violet  in  alkaline.  This  indicator  is  especially  suited 
for  the  determination  of  the  acidity  of  the  urine  by  titration. 

/C6H4OH 

7.  Phenolphthak'in,  C6H 


Add  4  drops  of  the  indicator  directly  to  the  solution. 

The  indicator  is  colorless  in  neutral  and  acid  reactions,  but 
becomes  red  in  the  presence  of  alkalies.  It  shows  the  pres- 
ence of  free,  combined,  mineral,  and  organic  acids,  and  acid 
salts  of  all  kinds.  It  therefore  gives  the  total  acidity.  It 
may  indicate  an  acid  reaction  when  dimethylaminoazoben- 
zene  shows  alkaline.  Explain  this. 

The  Inverting  Action  of  HCL     f 

To  5  c.c.  of  a  5  per  cent  cane-sugar  solution  add  5  c.c.  of 
1  per  cent  HC1  and  place  the  mixture  in  the  water-bath  for 
an  hour  at  40°  C.  At  the  end  of  this  time  cool,  neutralize, 
and  test  the  reducing  power  of  the  mixture.  Explain  the 
result. 

LACTIC  ACID,  CH3.CH(OH)-C^OH- 

Although  this  substance  is  commonly  considered  as  an 
abnormal  constituent  of  the  stomach  contents  it  may  exist 


GASTRIC  DIGESTION.  49 

as  a  normal  product  formed  during  the  digestion  of  a  meal 
rich  in  carbohydrates. 

The  following  experiments  are  designed  to  show  the  ordi- 
nary tests  for  lactic  acid  and  the  effect  upon  the  reliability 
of  these  tests,  of  the  simultaneous  presence  of  bodies  likely  to 
be  found  in  a  stomach  contents. 

(a)  To  successive  portions  of  5  c.c.  of  Uffelmaris  reagent 
add  a  few  drops  of  solutions  a,  c,  and  d,  under  Hydrochloric 
Acid.  Note  carefully  color  changes  and  make  deductions. 

Make  a  very  dilute  solution  of  Fe2Cl6  in  which  the  yellow 
color  is  hardly  visible.  Such  a  reagent  is  much  more  sensi- 
tive than  Uffelman's.  Use  5  c.c.  of  the  dilute  ferric  chloride 
solution  in  testing  each  of  the  following: 

(6)  Solutions  a,  c,  and  d  (under  HC1). 

(c)  A  solution  of  H2NaP04. 

(d)  Alcohol,  5  per  cent. 

(e)  A  1  per  cent  solution  of  saccharose;  glucose. 

Make  deductions  as  to  the  value  of  the  test  applied 
directly  to  gastric  contents. 

In  order  that  all  chances  for  error  may  be  avoided,  lactic 
acid  can  be  easily  separated  from  disturbing  conditions  by 
shaking  the  stomach  contents  or  gastric  juice  with  ether  in 
which  the  acid  is  soluble.  Such  an  ether  extract  is  evapo- 
rated carefully  on  the  water-bath,  the  residue  taken  up  with 
water  and  tested  with  the  dilute  Fe2Cl6  solution. 

As  this  ether  extract  cannot  contain  any  of  the  above 
substances,  the  presence  of  which  in  the  stomach  contents 
might  interfere  with  a  correct  diagnosis,  a  positive  test  for 
lactic  acid  in  this  case  is  decisive  evidence  of  its  presence. 

PEPSIN  AND  PEPSINOGEN. 

Pepsin  is  apparently  not  secreted  as  such,  but  appears 
first  in  the  gastric  mucosa  in  an  antecedent  form,  or  zymogen, 


50  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

pepsinogen.  This  latter  substance  shows  considerable  resist- 
ance to  reagents  (dilute  alkalies)  which  destroy  pepsin. 
Pepsinogen  is  itself  inactive  proteolytically,  but  seems  to  be 
converted  into  active  pepsin  by  the  action  of  the  HC1  of  the 
gastric  juice.  Pepsin  belongs  to  the  proteolytic  enzymes, 
converting  proteins  into  proteoses,  peptones,  amino  acids, 
and  diamines.  The  temperature  of  its  optimum  activity  is 
from  35-45°  C.  It  is  destroyed  at  65°  C. 

Pepsin  is  only  active  in  acid  solutions,  and  the  amount  of 
acidity  required  for  its  maximum  activity  varies  according  to 
the  character  of  the  acid  employed  and  the  form  of  protein 
to  be  digested.  Pepsin  is  easily  destroyed  by  weak  alkalies 
(0.01  per  cent),  and  less  readily  by  strong  acids  (5-10  per 
cent). 

The  following  set  of  experiments  should  prove  these 
facts: 

1.  A  glycerol  extract  of  a  pig's  gastric  mucosa  contains 
pepsinogen. 

2.  A  0.2%  HC1  extract  of  a  pig's  gastric  mucosa  contains 
pepsin  HCL 

3.  A  watery  extract  of  a  pig's  gastric  mucosa  contains 
pepsin. 

Make  use  of  the  above  extracts  numbered  1,  2,  and  3 
respectively,  and  in  each  test-tube  add  a  piece  of  fibrin, 
keeping  all  at  40°  C.  in  the  water-bath. 

(a)  Fibrin  +  5  c.c.  of  0.2%  HC1. 

(b)  "      +5  c.c.  of  solution  3. 

(c)  "     -f  one  drop  of  solution  1  +  5  c.c.  of  water. 

(d)  "      +  5  c.c.  of  solution  2. 

(e)  "      +  one  drop  of  solution  1  +  5  c.c.  of  0.2%  HC1. 
(/)        "      +  5  c.c.  of  solution  2  (the   latter  having  pre- 
viously been  heated  to  boiling  and  again  cooled). 

(#)  Fibrin  +  one  drop  of  solution  1  (the  latter  having 


GASTRIC  DIGESTION.  51 

previously  been  heated  to  boiling  with  5  c.c.  water  and  again 
cooled)  +  5  c.c.  of  0.2%  HC1. 

(h)  Fibrin  +  5  c.c.  of  solution  3  +  5  c.c.  of  0.8%  lactic 
acid. 

(&)  Fibrin  +  5  c.c.  of  solution  3  +  5  c.c.  of  1%  oxalic  acid. 

(ro)      "      +5  "    "        "       3  +  5  "    "5%HC1. 

(n)      "      +5"    "        "       3  +  5"    "0.5%Na,CO,. 

(p)      "      +5  "    "        "       3  +  3  "    "bile. 

Note  carefully,  in  each  case,  the  relative  rapidity  with 
which  the  flock  of  fibrin  is  disintegrated. 


PEPTIC   PROTEOLYSIS. 

The  previous  experiments  have  indicated  that  the  action 
of  pepsin  is  directed  toward  the  transformation  of  the  proteins 
by  a  process  of  cleavage  into  soluble  and  diffusible  products. 
The  question  concerning  the  characterization,  separation, 
and  identification  of  these  soluble  digestive  products  appears 
at  present  to  rest  in  a  state  of  uncertainty,  but  in  a  general 
way  they  are  conventionally  divided  into  the  proteoses,  pep- 
tones, and  a  mixture  of  nitrogenous  substances  ("amino" 
bodies)  only  characterized  by  not  giving  the  biuret  reaction. 
The  current  method  for  the  separation  of  these  bodies  is 
based,  first,  upon  the  precipitation  of  the  proteoses  by  com- 
plete saturation  of  their  solution  with  (NH4)2S04,  and,  second, 
upon  the  precipitation  of  the  peptones  in  the  filtrate  (from 
the  proteoses)  by  means  of  iodo-potassium  iodide,,  The 
remaining  substances  are  removed  from  their  solution 
by  the  addition  of  phosphotungstic  acid.  The  proteoses 
allow  of  a  further  separation  by  means  of  fractional  precipi- 
tation with  (NHJ2S04,  and  the  peptones  are  divided  into 
two  fractions  by  95  per  cent  alcohol.  This  method  isolates 


52  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

three  fractions  of  proteoses  and  two  of  peptones,  the  pro- 
cedure for  which  is  the  following: 

For  the  purposes  of  study,  a  pepsin-hydrochloric  acid 
digestion  of  meat  or  fibrin  should  be  prepared,  using  0.3% 
HC1  and  0.5  grm.  pepsin  per  liter  (Grubler's  purissimum  or 
Parke-Davis'  scale  pepsin).  Allow  the  digestion  to  proceed 
at  40°  C.  for  three  days.  After  filtration  of  the  digestion 
mixture  and  exact  neutralization  with  dilute  NaOH  the 
solution  is  ready  for  use.  Heat  a  little  of  it  to  boiling  to 
show  that  no  coagulable  proteins  exist  in  the  mixture. 

To  a  given  quantity  of  the  digestion  mixture  add  an 
equal  volume  of  a  saturated  solution  of  (NH4)2S04.  Stir 
the  solutions  together  and  allow  the  mixture  to  stand  until 
the  resulting  precipitate  has  settled  sufficiently  to  allow 
of  considerable  decantation.  Filter  the  remainder.  This 
precipitate  obtained  by  half  saturation  of  the  mixture  repre- 
sents 

Fraction  I. — It  may  be  further  separated  into  two  parts: 
one  soluble  in  95  per  cent  alcohol — protoproteose, 
and  the  other  remaining  insoluble — heteroproteose. 

In  the  constitution  of  protoproteose  the  aromatic 
groups  predominate,  while  in  heteroproteose  the 
fatty  acid  radicals  are  chiefly  found. 

To  the  filtrate  from  Fraction  I  add  one-half  its  volume  of 
a  saturated  solution  of  (NH4)2S04.  Allow  this  to  stand 
and  proceed  as  before.  This  second  precipitate  obtained 
by  two-thirds  saturation  corresponds  to 

Fraction  II. — It  consists  of  Deuteroproteose.  A  and  contains 
the  greater  part  of  the  lead-blackening  sulphur 
which  was  present  in  the  original  proteid  molecule. 
On  this  account  it  may  be  termed  Thioproteose. 

Completely  saturate  the  filtrate  from  Fraction  II  with 


GASTRIC  DIGESTION.  53 

(NH4)2S04  (in  substance),  allow  the  mixture  to  stand,  and 
treat  as  previously. 

Fraction  III. — This  precipitate  is  termed  Deuteroproteose  B  or 
Synproteose.  It  contains  the  greater  part  of  the  car- 
bohydrate nucleus  of  the  original  protein  molecule 
and  is,  therefore,  sometimes  called  Glycoproteose. 

To  the  filtrate  from  Fraction  III  add  TV  its  volume  of  a 

N 

—  H2S04  solution,  which  is  saturated  with  (NH4)2S04.     Pro- 
ceed with  the  precipitation  and  filtration  as  before. 

Fraction  IV. — This  fraction,  which  in  the  case  of  some 
proteins  is  absent,  is  designated  as  Deuteroproteose  C. 

The  acid-saturaled  filtrate  from  Fraction  IV  is  treated 
with  a  solution  of  iodo-potassium  iodide  (saturated  with 
(NH4)2SO4)  until  no  further  precipitate  results.  This  is 
allowed  to  settle  and  is  then  filtered  off.  This  precipitate 
upon  treatment  with  95  per  cent  alcohol  subdivides  into  a 
soluble  and  insoluble  fraction.  If  the  insoluble  residue 
is  filtered  off  it  forms 

Fraction  V,  denoted  as  Peptone  A;  and 

Fraction  VI,  which  consists  of  the  alcoholic  filtrate  from 
Fraction  V,  is  called  Peptone  B. 

Very  little  is  known  regarding  the  character  of 
these  two  peptone  fractions.  Undoubtedly  they 
contain  peptides  of  varying  complexity  mixed  with 
amino  acids.  Fraction  V  gives  the  biuret  reaction 
while  Fraction  VI  does  not;  hence  the  former  frac- 
tion must  possess  the  most  complex  polypeptides. 

The  "amino"  bodies  are  precipitable  with  phospho- 
tungstic  acid  and  remain  in  greater  part  in  the  filtrate  sub- 
sequent to  the  treatment  with  iodo-potassium  iodide. 


54  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

More  or  less  of  acid  albuminate  may  be  formed  during 
the  initial  stage  of  the  digestion  (first  half -hour),  but  this 
quickly  disappears  and  apparently  does  not  participate 
in  the  future  formation  of  the  proteoses,  peptones,  and 
"amino"  bodies. 

Perform  the  following  tests  with  the  different  fractions: 

(a)  Try  the  biuret  reaction  on  each  fraction. 

(6)  Test  Fractions  I  and  //  for  lead-blackening  sulphur. 

(c)  Boil  Fraction  III  with  10  per  cent  H2S04  for  an  hour. 
Cool,  neutralize,  and  test  the  mixture  with  Fehling's  solu- 
tion. 

(d)  Show,  first,  that  the  solutions  of  Fractions  I,  II,  IIIt 
IV  are  precipitated  by  nitric,  picric,  and  trichloracetic  acids, 
and  that  the  precipitates  so  produced  disappear  when  heat 
is  applied  and  reappear  upon  cooling;    and,  second,  that 
they  are  non-coagulable  by  heat  and  respond  to  the  acetic 
acid  and  potassium  ferrocyanide  reaction. 

(e)  Prove  that  the  peptones  (Fractions  V  and  VI)   are 
not  precipitated  by  nitric  or  trichloracetic  acids;  that  Millon's 
and  xanthoproteic  reactions  give  indecisive  results;    that 
lead-blackening  sulphur  is  absent;  and  that  both  tannic  and 
picric  acids  combine  to  form  insoluble  bodies. 

It  must  be  finally  emphasized  that  these  various  fractions 
are  not  to  be  considered  as  unit  substances.  The  method  of 
separation  just  outlined  simply  attempts  to  isolate  certain 
mixtures  ("fractions")  each  of  which  contains  in  greater 
part  some  characteristic  component  group  or  complex 
broken  off  during  the  peptic  cleavage  of  the  original  protein 
molecule.  Thus  in  Deuteroproteose  A  the  lead-blackening 
or  cystine  nucleus  predominates,  while  Deuteroproteose  B 
is  characterized  by  containing  the  larger  part  of  the  carbohy- 
drate complex. 


GASTRIC  DIGESTION.  55 

RENNIN  OR  CHYMOSIN. 

This  body  is  classed  as  a  coagulating  enzyme  acting 
upon  caseinogen  with  the  formation  of  an  albumose-like 
body  and  soluble  casein.  The  latter  in  the  presence  of  cal- 
cium salts  is  precipitated  in  the  form  of  a  coagulum  or  curd 
which  is  termed  (insoluble)  casein. 

Rennin  is  affected  by  varying  conditions  (temperature, 
reaction,  etc.)  in  a  way  similar  to  pepsin. 

Make  use  of  the  rennin  solution  given  and  prepare  five 
test-tube  mixtures  as  follows,  using  in  each  case  about  5  c.c. 
of  milk  and  holding  them  at  40°  C.  for  fifteen  minutes; 
in  each  experiment  add  the  rennin  solution  last. 

(a)  Milk +  15  drops  of  0.3%  HC1. 

(6)      "    +1  c.c.  of  rennin  solution. 

(c)  "    +1   "    "       "  "        +2    cc.    ammonium 
oxalate. 

(d)  Milk  +  2  c.c.  of  rennin   solution  +  5  drops  0.3% 
HC1. 

(e)  Milk  +  2   c.c.   of  rennin  solution  +  10  drops  0.5% 
Na2C03. 

Note  the  results  carefully.  If  (c)  has  not  clotted,  heat 
it  to  boiling  to  kill  the  enzyme.  Cool  and  add  a  few  drops 
of  calcium  chloride  solution.  What  is  this  precipitate 
which  settles  out?  What  are  the  effects  of  acids  and  alka- 
lies on  the  milk  alone?  Why  did  not  the  milk  clot  in  experi- 
ment c? 


PANCREATIC  DIGESTION. 

The  composition  and  character  of  pancreatic  juice  varies 
greatly  under  different  conditions.  Thus  far  it  has  been 
practically  impossible  to  collect  the  juice  in  a  manner  which 
will  permit  of  the  assumption  that  it  is  comparable  to  that 
secreted  into  the  intestine. 

The  juice  as  obtained  from  a  temporary  fistula  is  clear, 
viscous,  and  alkaline  in  reaction,  the  alkalinity  corresponding 
to  about  a  0.5  per  cent  Na2C03  solution.  It  differs  from 
the  gastric  juice  hi  being  rich  in  proteins,  having  a  sp.  gr. 
of  about  1.030.  The  character  and  quantity  of  the  enzymes 
which  may  be  present  hi  the  fluid  varies  considerably  accord  - 
ing  to  the  nature  of  the  diet. 

The  following  have  been  shown  to  exist  in  different 
extracts  of  the  gland  and  in  the  secretion  itself  at  certain 
times. 

1.  Trypsin — a  proteolytic  enzyme  appearing  first  in  the 
gland  as  a  zymogen,  trypsinogen,  entirely  comparable  to 
pepsinogen. 

2..Amylopsin  or  pancreatic  diastase — an  amylolytic  en- 
zyme almost  identical  with  ptyalin. 

3.  Steapsin  or  lipase — a  lipolytic  enzyme  probably  not 
characteristic  of  the  pancreatic  juice. 

4.  Pancreatic  rennin — a  coagulating  enzyme  acting  on 
the  caseinogen  of  milk  in  a  way  somewhat  similar  to  the 
gastric  rennin.     Various  extracts  of  the  pancreatic  gland 

56 


PANCREATIC  DIGESTION.  57 

have  been  prepared  each  with  a  view  to  obtaining  predominat- 
ing reactions  for  the  specific  enzyme.     (See  Appendix.) 

TRYPSIN  AND  TRYPSINOGEN. 

Trypsin  is  active  in  an  alkaline,  faintly  acid  or  neutral 
solution,  but  its  maximum  reaction  occurs  in  a  1  per  cent 
solution  of  Na2C03.  It  is  killed  at  a  temperature  about 
55°  C. 

Use  the  proteolytically  active  pancreatic  extract  and 
prepare  test-tube  digestions  in  the  water-bath  at  40°  C.,  as 
under  gastric  digestion. 

(a)  Fibrin  +  5  c.c.  of  the  pancreatic  extract. 

(6)  "  +5"  "  "  "  "  +  2  c.c.  0.5% 

Na2C03. 

Compare  the  manner  of  action  of  the  trypsin  upon  the 
fibrin  to  that  of  the  pepsin. 

(c)  Fibrin  +  5  c.c.  of  0.5%  Na2CO3. 

(d)  "     +  5  "    "    "  "      li       +5  c.c.  pancreatic  ex- 
tract which  has  been  previously  boiled  and  cooled. 

TRYPTIC   PROTEOLYSIS. 

As  regards  its  proteolytic  activity,  the  enzyme  trypsin 
is  more  energetic  and  far-reaching  than  pepsin.  Conse- 
quently in  pancreatic  digestion  there  takes  place  a  more 
rapid  and  deep-seated  cleavage  of  the  protein  molecule 
than  is  possible  in  the  stomach.  Proteoses  and  peptones 
are  formed  in  the  middle  stages  of  the  digestion,  but  these 
are  rapidly  carried  over  into  substances  with  a  relatively 
small  molecular  weight  which  do  not  give  the  biuret  reaction 
and  consequently  cannot  be  termed  peptones.  It  is  now 
considered  that  the  end  products  of  a  long-continued  tryptic 
digestion  of  the  ordinary  proteins  comprise  the  amino-acids, 


58    LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

viz.,  leucine,  tyrosine,  aspartic  and  glutamic  acids;  the  hexone 
bases,  viz.,  arginine,  lysine,  and  histidine;  the  so-called  trypto- 
phane  or  proteinochromogen,  which  is  an  indole  derivative  ;  and 
probably  other  bodies  whose  nature  is  not  well  understood. 

Prepare  a  pancreatic  digestion  as  follows: 

Place  in  a  large  flask  (5-10  liters)  500-1000  grms.  of 
fibrin,  a  liter  of  an  infusion  of  Kuhne's  dried  pancreas,  and 
1000-2000  c.c.  of  0.25  per  cent  Na2C03;  add  plenty  of  chloro- 
form or  powdered  thymol,  and  allow  the  mixture  to  digest  at 
40°  C.  for  at  least  a  week. 

At  the  end  of  this  time  the  solution  is  exactly  neutralized 
with  dilute  H2S04  and  concentrated  to  about  one-half  its 
volume;  it  is  then  filtered.  From  the  filtrate  the  proteoses 
are  removed  by  saturation  with  (NH4)2S04  in  a  nearly 
boiling  solution.  These  are  filtered  off  and  tho  filtrate  still 
further  concentrated.  During  this  procedure  relatively 
large  quantities  of  leucine  and  tyrosine  should  crystallize  out 
of  the  mixture  along  with  the  (NH4)2S04.  These  must  be 
filtered  off  and  the  (NH4)2S04  removed  from  the  filtrate  by 
means  of  Ba(OH)2  and  BaC03.  The  BaS04  is  again  re- 
moved by  filtration  and  to  the  new  filtrate  dilute  H2S04 
added  to  just  precipitate  the  excess  of  Ba  in  the  solution. 
After  filtration  the  solution  will  contain  the  hexone  bases. 

Compare  the  quantities  of  proteoses  and  peptones  ob- 
tained in  this  digestion  with  those  from  the  peptic  proteolysis. 


TYROSINE,  CH2-CH-COOH. 


Use  the  crystals  obtained  in  the  pancreatic  digestion. 

(a)  Examine  them  under  the  microscope. 

(6)  Try  to  dissolve  some  in  cold,  then  warm  water.     Then 


PANCREATIC  DIGESTION.  59 

add  about  5  c.c.  of  Millon's  reagent  to  the  test-tube  and 
heat.     Why  is  this  result  positive? 

(c)  Piria's  Test.  —  Place  a  few  of  the  tyrosine  crystals  upon 
a  small  watch-glass  with  two  drops  of  concentrated  H2S04  and 
warm  for  half  an  hour  on  the  water-bath.     Then  transfer  it 
with  about  15  c.c.  water  to  a  test-tube  and  neutralize  with 
BaC03  in  substance.     Filter  and  add  a  few  drops  of  weak 
ferric  chloride  solution  (neutral).    A  positive  test  is  evidenced 
by  the  formation  of  a  violet  .color. 

(d)  Morner's  Test.  —  To  a  little  of  the  powder  placed  in  a 
test-tube  add  a  few  c.c.  of  Morner's  reagent,  and  heat  care- 
fully to  boiling.     A  green  color  develops  which  is  quite 
lasting. 

CHJX  /NH* 

LEUCINE,          >CH-CH,-CH     „ 

PTT  /  \          /}J 


X)H 

Make  use  of  the  substance  obtained  from  the  pancreatic 
digestion. 

(a)  Examine  some  crystals  under  the  microscope. 

(6)  Place  a  little  in  a  clean  dry  test-tube  and  warm  care- 
fully. The  leucine  sublimes,  without  melting,  on  the  cold 
parts  of  the  tube.  If  the  powder  is  heated  too  high  or 
suddenly,  the  substance  is  decomposed  and  the  odor  of 
amylamine  is  given  off.  Write  the  equation  for  the  formation 
of  this  substance. 

(c)  Scherer's  Test.  —  Heat  a  little  leucine  with  two  drops  of 
concentrated  HN03  upon  a  platinum  foil  over  a  free  flame 
until  a  colorless  residue  is  obtained.  Then  add  two  drops 
of  NaOH  and  evaporate  carefully  in  the  flame.  The  mass 
becomes  dark  red  in  color  and  rolls  around  on  the  foil  like 
an  oil-drop. 


60  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 


AMYLOLYSIS. 

Amylopsin  is  stronger  in  its  action  than  ptyalin;  other- 
wise the  conditions  which  influence  its  activity  are  practically 
the  same  as  the  salivary  diastase. 

Make  use  of  the  glycerol  or  alcoholic  extract  of  the  pan- 
creas. 

To  10  c.c.  of  starch  paste  add  5  c.c.  of  pancreatic  extract 
and  place  in  the  water-bath  at  40°  C.  Test  from  time  to 
time  with  the  iodine  solution  as  you  did  under  Salivary 
Digestion.  Do  you  get  an  achromic  point?  Finally  test  for 
a  reducing  sugar.  What  is  the  character  of  this? 

LIPOLYSIS. 

Steapsin  is  the  least  stable  of  all  the  pancreatic  enzymes, 
and  is  particularly  sensitive  to  the  action  of  acids,  being 
destroyed  easily  by  all  except  the  higher  fatty  acids. 

Its  action  on  neutral  fats  tends  toward  a  breaking  down 
of  the  fat  molecule  into  fatty  acids  and  glycerol. 

For  the  following  experiments  use  the  alkaline  glycerol 
extract  of  the  gland. 

(a)  To  10  c.c.  of  "litmus  milk"  add  5  c.c.  of  the  pan- 
creatic extract.  Place  the  test-tube  in  the  water-bath  at 
40°  C.  What  is  the  cause  of  the  change  which  takes  place? 

(6)  Repeat  experiment  (a)  with  the  exception  that 
boiled  pancreatic  extract  is  used.  Note  any  change? 

(c)  Instead  of  "litmus  milk,"  try  the  experiment  with 
neutral  ethyl  butyrate.  What  is  the  equation  for  the  reac- 
tion which  takes  place? 


PANCREATIC  DIGESTION.  61 


PANCREATIC  RENNIN. 

The  ability  of  pancreatic  juice  to  cause  the  clotting  of 
milk  has  long  been  known;  but  only  of  late  has  the  action 
been  ascribed  to  a  definite  enzyme,  which,  on  account  of  the 
similarity  in  its  function  to  the  milk-clotting  enzyme  of  the 
stomach,  has  been  called  pancreatic  rennin.  This  sub- 
stance also  possesses  the  power  of  bringing  about  a  peculiar 
changed  condition  of  the  caseinogen  of  the  milk  by  which 
the  protein  is  rendered  liable  to  coagulation.  The  coagu- 
lable  protein  is  termed  metacasein,  and  its  appearance  is 
denoted  as  the  metacasein  reaction.  Metacasein  is  appar- 
ently an  intermediate  stage  in  the  action  of  the  pancreatic 
rennin  upon  caseinogen,  in  which  pancreatic  casein  is  the 
end  product.  Pancreatic  casein  differs  in  some  slight  degree 
from  true  casein. 

(a)  To  5  c.c.  of  milk  add  1  c.c.  of  the  pancreatic  extract 
and  place  the  mixture  at  40°  C.  Note  the  formation  of  the 
clot. 

(6)  To  10  c.c.  of  the  milk  add  1  c.c.  of  the  pancreatic  ex- 
tract diluted  with  2  volumes  of  water.  Place  the  test-tube 
at  40°  C.  and  from  time  to  time  remove  portions  (1  c.c.)  of 
the  mixture  and  heat  each  to  boiling.  In  one  of  them 
coagulation  in  fine  flakes  will  occur.  Note  the  time  from  the 
beginning  of  the  experiment. 

(c)  Make  an  exact  duplicate  of  the  preceding  experi- 
ment, and  at  the  time  at  which  the  appearance  of  the  meta- 
casein reaction  would  be  expected,  judging  from  the  last 
experiment,  take  out  the  tube  from  the  bath.  Examine 
a  drop  under  the  microscope.  Stand  the  tube  in  a  cool 
place  and  note  the  formation  of  a  clot.  This  will  dissolve 
when  replaced  in  the  water-bath. 


INTESTINAL  PUTREFACTION. 

The  chief  interest  in  the  study  of  the  effects  produced 
by  bacteria  upon  protein  matter  lies  in  the  direction  of  the 
additional  insight  which  can  be  obtained  into  the  consti- 
tution of  the  protein  molecule.  This  becomes  possible 
.since  the  microorganisms  effect  a  cleavage  of  the  mole- 
cule which,  while  similar  in  character  to  that  exerted  by 
the  digestive  enzyme,  is  more  vigorous  and  deep-seated 
than  even  trypsin  itself;  and  the  products  obtained  are  so 
simple  in  composition  and  structure  that  they  have  yielded 
easily  to  investigation. 

The  most  important  substances  which  result  from  bac- 
terial decomposition  of  proteins  may  be  divided  as  follows : 

1.  Tyrosine  and  its  derivatives,  the  phenols,  aromatic 
acids,    and    oxy-acids    (e.g.,  oxyphenylpropionic  and  oxy- 
phenylacetic  acids). 

2.  Indole  and  its  derivatives,   skatole,  skatole-carbonic 
acid,  and  tryptophane. 

3.  Fatty  acids  and  derivatives,  leucine,  etc. 

4.  Sulphur    derivatives   and   gases,    hydrogen   sulphide, 
methyl  mercaptan,  CEU,  CC>2,  and  NHs. 

5.  Proteoses    and    "  peptones "    are    present    in    small 
amounts  as  intermediary  products. 

The  following  method  is  employed  for  the  preparation 
and  separation  of  the  various  products.  It  lends  itself  also 
for  demonstration  purposes,  and  the  different  distillates 
can  be  studied  individually. 

62 


INTESTINAL  PUTREFACTION.  63 

Prepare  a  mixture  of  lean  chopped  beef  and  coagulated 
egg-white  in  equal  amounts,  and  add  thereto  about  1  liter 
of  water  to  each  pound  of  material  employed. 

If  cultures  of  colon  bacillus  are  available,  sterilize  the 
flask  and  its  contents  and  add  some  of  the  culture.  Other- 
wise make  the  mixture  alkaline  (add  to  each  liter  100  c.c. 
of  10%  Na2C03),  and  inoculate  with  some  meat  which  has 
been  allowed  to  putrefy.  After  shaking  thoroughly  stop  the 
flask  with  a  bored  cork  which  is  provided  with  a  glass  tube 
to  which  is  attached  a  valve  (wash-bottle)  of  mercuric 
cyanide  (3  per  cent).  This  serves  to  collect  the  methyl 
mercaptan  and  to  render  the  flask  less  odorous.  Allow  the 
flask  to  remain  at  40°  C.  for  two  to  three  weeks.  The 
separation  of  the  products  takes  place  as  follows: 

Distil  the  putrefactive  mixture  with  steam  until  the 
distillate  equals  one-half  the  volume  of  the  original  mixture. 
There  is  thus  provided,  1,  a  distillate  (A) ;  and,  2,  a  distil- 
lation residue  (A),  which  will  be  treated  separately. 

Distillate  A. 

Acidify  with  weak  HC1  and  redistil  (usually  one-half  volume). 


Distill 


llate  B.  Distillation-residue  B. 

Make  alkaline  with  (NH4C1.) 

KOH  and  again  distil. 

Distillate  C.  Distillation-residue  C. 

Keep  the  first  few  c.c.  Saturate  with  CO,  gas 

of  this  distillation  sep-  and  redistil, 

arate    from    the    rest 
(Indole  and  Skatole). 


ite  D.  Distillation-residue  D. 

(Phenols.)  (Fatty  adds.) 


64  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 
Distillation-residue  B. 

Upon  evaporation  this  can  be  shown  to  contain  NH4C1 
formed  from  the  NH3  of  the  putrefaction  product  and  the 
HC1  added  in  the  manipulation. 

Distillate  C. 

Contains  indole  and  skatoie  (see  Conjugate  Sulphates). 

The  first  distillation  carries  over  skatoie;  this  may  be 
tested  as  follows: 

(a)  To  2  c.c.  add  a  drop  of  HNO3  and  a  drop  or  two  of 
KN02  solution  (2  per  cent).  Notice  the  turbidity. 

(6)  Add  some  concentrated  HC1  to  some  of  the  distillate. 

Try  the  succeeding  tests  with  the  main  distillate  (indole}. 

(a)  Moisten  a  piece  of  pine  wood  with  concentrated  HC1 
and  dip  it  into  the  distillate.  A  cherry-red  color  appears 
on  the  wood. 

(6)  Legal's  reaction. — To  a  few  c.c.  of  the  distillate  add 
5  drops  of  a  freshly  prepared  solution  of  sodium  nitroprusside ; 
then  make  it  alkaline  with  2  drops  of  NaOH.  A  violet  color 
results,  which  resolves  itself  into  deep  blue  upon  acidifica- 
tion with  glacial  acetic  acid. 

(c)  Acidify  the  distillate  with  HN03  and  add  2  drops 
of  KNO2  solution  (see  Skatoie).  A  red  precipitate  of  nitroso- 
indole  nitrate  results. 

Distillate  D. 

Contains  phenol  and  p.cresol.  (Make  very  faintly  alkaline 
with  KOH  and  concentrate  to  a  small  volume.) 

For  the  reactions  of  phenol  see  p.  96. 

p.cresol  gives  with  ferric  chloride  solution  a  dirty-green 
coloration. 


INTESTINAL  PUTREFACTION.  65 


Distillation-residue  D. 

Acidify  this  with  concentrated  HC1  and  shake  it  out  with 
a  small  amount  of  ether.  Upon  evaporation  of  the  ether 
extract,  the  volatile  fatty  acids  remain. 

Distillation-residue  A. 
Concentrate  and  filter;  then  extract  with  ether. 

r~  ""i 

Ether  extract.  Residual  solution. 

Remove  the  ether  by  evaporation  Concentrate  until  crys- 

and    extract    the    residue    with  tallization  begins;  filter, 
warm  water;  filter. 


Crystalline  precipitate. 
(Leucine  and  tyrosine.) 

Filtrate  B. 
/Proteoses,  peptonesA 
1  tryptophane    and      I 
\aromatic  acids.          / 

1                1 

rate  A,                Residue. 

(Oxy-acids  and  skatole- 
carbonic  acid.) 

(non- volatile  fatty  acids). 

Filtrate  A. 

This  contains  p-oxyphenylacetic  acid,  p-oxyphenylpro- 
pionic  acid,  and  skatole-carbonic  acid. 

(a)  Try  the  ferric  chloride  reaction  (see  Phenol) . 

(6)  Try  Millon's  reaction.  Which  of  the  three  sub- 
stances are  these  tests  for? 

(c)  Perform  test  (c)  under  Indole  and  compare  the  result- 
ing colors  and  precipitates  with  those  for  indole  and  skatole. 

* 

Residue. 
See  Fatty  Acids  for  tests. 


66  LABORATORY  WORK  L\  PHYSIOLOGICAL  CHEMISTRY. 

Crystalline  Precipitate. 
See  Leucine  and  Tyrosine. 

Filtrate  B. 

See  Proteoses  and  "Peptones." 

Tryptophane,  indoleaminopropionic  acid  (proteinochro- 
mogen).  This  substance  is  formed  as  one  of  the  products  in 
the  pronounced  cleavage  of  proteins  as  in  tryptic  digestion 
and  putrefaction.  Its  presence  in  the  protein  molecule  is  of 
considerable  importance  to  the  animal  economy.  It  is  sup- 
posed to  stand  as  the  mother  substance  of  the  various  pig- 
ments of  the  body.  Add  a  few  c.c.  of  chlorine  or  bromine 
water  to  the  solution;  a  reddish-violet  precipitate  results 
(proteinochrome). 

Finally  examine  the  mercuric  cyanide  valve  which  was 
attached  to  the  flask  containing  the  putrefactive  mixture. 

Hydrogen  sulphide  has  produced  a  black  precipitate  of 
HgS. 

Methyl  mercaptan  is  indicated  by  the  presence  of  a  gray- 
ish-green precipitate. 


BILE. 

The  function  of  the  bile  is  mainly  excretory  in  character, 
consisting  in  the  removal  by  way  of  the  intestine  of  various 
relatively  insoluble  substances,  such  as  cholesterol  and  leci- 
thin. Secondarily,  it  exerts  an  emulsifying  power  upon 
the  fats,  and  fat  absorption  is  markedly  decreased  in  its 
absence.  In  this  sense  it  may  be  classed  under  the  digestive 
juices. 

Bile  as  it  is  secreted  by  the  liver  forms  a  clear,  limpid 
solution  of  a  color  either  yellowish-red,  brown,  or  green  ac- 
cording to  the  species  of  animal  from  which  it  is  obtained. 
It  reacts  alkaline  to  litmus,  but  acid  to  phenolphthalein  and 
possesses  a  decidedly  bitter  taste  and  an  odor  of  musk.  By 
its  stay  in  the  gall-bladder  the  character  of  the  fluid  is 
changed  considerably.  Absorption  of  water  and  admix- 
ture of  mucus-like  substances  derived  from  the  walls  of  the 
bladder  increase  the  specific  gravity  from  1.010  to  1.035. 
When  taken  from  the  gall-bladder,  bile,  therefore,  presents 
a  ropy,  viscous  appearance  and  contains  about  10  per  cent 
of  solids.  Bile  holds  the  following  substances  in  solution: 
the  salts  of  the  bile-acids,  the  bile-pigments,  mucin  or  phospho- 
protein,  cholesterol,  lecithin,  inorganic  salts,  and  traces  of 
fat,  soaps,  and  urea. 

Note  the  color,  consistency,  and  reaction  of  the  samples 
presented. 

Notice  the  differences  in  color  between  the  ox  and  dog 

bile. 

67 


68  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY- 

Use  the  diluted  bile  for  the  following  reactions: 

(a)  Add  acetic  acid  drop  by  drop  to  5  c.c.  of  the  bile. 

Is  this  precipitate  mucin?     How  could  you  prove  it?    Filter 

and  test  the  filtrate  for  protein.     What  is  the  most  suitable 

test? 

(6)  To  1  c.c.  of  olive-oil  add  10  c.c.  of  water  and  shake 

thoroughly.    Allow  it  to  stand  in  the  rack. 

(c)  To  1  c.c.  of  olive-oil  add  10  c.c.  of  diluted  bile.     Allow 
this  to  stand.     Compare  the  permanency  of  the  emulsion. 

(d)  To  10  c.c.  of  a  gastric  digestion  mixture  add  diluted 
bile,  drop  by  drop.    What  is  this  precipitate  and  its  signifi- 
cance? 

CONJUGATE  BILE  ACIDS. 

These  are  present  as  the  sodium  salts  of  taurocholic  and 
glycocholic  acids,  and  are  formed  by  the  combination  of 
cholalic  acid  with  taurin  and  glycocoll  respectively.  Tauro- 
cholic acid,  by  virtue  of  the  taurin,  contains  sulphur  and 
has  a  solvent  action  on  the  more  insoluble  glycocholic  acid. 
It  is  more  abundant  in  the  bile  of  carnivora,  while  glyco- 
cholic acid  predominates  in  that  of  man  and  herbivora. 
Together  they  tend  toward  rendering  the  cholesterol  and 
lecithin  more  soluble  and  exert  a  distinctly  inhibitory  action 
on  the  heart,  slowing  its  rhythm. 

Pettenkofer's  Test. — Place  5  c.c.  of  concentrated  H2S04  in 
a  clean  dry  test-tube.  In  another  test-tube  place  5  c.c.  of 
diluted  bile  to  which  has  been  added  a  few  drops  of  a  2  per 
cent  cane-sugar  solution  or  a  solution  of  furfurol  1 : 1000.  Pour 
the  diluted  bile  carefully  down  the  sides  of  the  tube  con- 
taining the  H2S04  so  that  the  two  fluids  do  not  mix.  (Method 
of  stratification.)  Notice  the  coloration  at  the  line  of  con- 
tact of  the  two  solutions.  Shake  the  tube  slightly,  allowing 
a  little  more  of  the  bile  to  come  in  contact  with  the  H2S04. 


BILE.  69 

The  temperature  must  never  rise  above  70°  C;  to  avoid 
this,  cool  the  tube  under  the  tap.  Upon  careful  mixing 
and  cooling  as  described  above,  the  whole  solution  finally 
becomes  cherry-red  or  reddish  purple.  Such  a  solution 
shows  a  definite  and  characteristic  spectrum  which  distin- 
guishes it  from  other  substances,  giving  the  same  reaction, 
such  as  phenol,  petroleum,  fusel-oils,  pyrocatechinol,  choles- 
terol, and  proteins. 

Crystallization  of  the  Bile  Salts  (Phttner's}. 

Mix  20  c.c.  of  the  bile  with  sufficient  animal  charcoal 
to  form  a  thick  paste  and  allow  the  mass  to  evaporate  on 
the  water-bath  to  dryness.  Grind  up  the  dry  residue  and 
extract  it  in  a  flask  with  25  c.c.  of  absolute  alcohol  on  the 
water-bath  for  15-20  minutes.  Filter  and  to  the  filtrate  add 
ether  until  a  slight  precipitate  is  visible.  Cover  the  vessel  and 
set  it  away  for  a  few  days.  Examine  the  crystals.  Dissolve 
some  in  alcohol  and  try  Pettenkofer's  Test. 

BILE  PIGMENTS. 

The  differences  in  color  which  were  noticed  in  the  various 
samples  of  bile  are  dependent  upon  the  presence  in  pre- 
dominating amounts  of  certain  pigments  of  which  the  two 
most  important  are  bilirubin  and  biliverdin.  The  former 
is  chiefly  present  in  the  bile  of  carnivora,  while  that  of  the 
herbivora  contains  biliverdin  in  greater  quantities.  The 
bile  pigments  are  closely  connected  generically  and  probably 
chemically  with  the  blood  and  urinary  pigments. 

Bilirubin  is  insoluble  in  water,  somewhat  soluble  in  alcohol 
and  ether,  and  dissolves  readily  in  chloroform,  benzene,  and 
acids  and  alkalies.  It  oxidizes  in  the  air  to  biliverdin. 

Biliverdin  is  soluble  in  alcohol,  partly  soluble  in  ether, 
and  insoluble  in  chloroform  and  water. 


70  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

The  pigments  possess  the  chemical  characteristics  of 
weak  acids  and  are  present  in  biliary  calculi  as  calcium  salts. 

Use  diluted  bile  for  the  following  tests: 

Gmelin's  Test. — Stratify  5  c.c.  of  yellow  HN03  and  5  c.c. 
of  diluted  bile  as  explained  in  the  previous  experiment. 
Notice  the  play  of  colors  at  the  junction  of  the  two  liquids 
— green,  then  blue,  violet,  red,  and  yellow.  To  what  are 
these  colors  due,  and  what  specific  substances  do  they  indi- 
cate? 

Smith's  Test. — Stratify  5  c.c.  of  diluted  bile  and  3  c.c.  of 
tincture  of  iodine.  Notice  the  bright  green  ring. 

Huppert's  Test.— To  10  c.c.  of  bile  add  an  equal  volume 
of  milk  of  lime  and  shake  thoroughly.  Filter  off  and  after 
washing  once  with  water  remove  the  precipitate  to  a  small 
beaker  with  25  c.c.  of  alcohol  acidulated  with  a  few  drops  of 
HC1.  Warm  the  beaker  in  a  water-bath  until  the  alcohol 
begins  to  assume  an  emerald-green  color.  What  is  the  chem- 
istry of  this  reaction? 

Hammarsten's  Test. — To  5  c.c.  of  Hammarsten's  reagent 
add  five  drops  of  diluted  bile.  A  green  color  immediately 
develops.  Upon  the  further  addition  of  the  reagent  in  small 
quantities  to  the  mixture,  the  same  play  of  colors  may  be 
obtained  as  in  Gmelin's  test. 

Analysis  of  a  Biliary  Calculus. 

Place  some  of  the  pulverized  calculus  in  a  clean  dry  test- 
tube  with  15  c.c.  of  ether.  Shake  thoroughly.  Filter  off  the 
ether  through  a  dry  filter  and  funnel  into  a  porcelain  dish 
and  allow  it  to  evaporate  in  the  air.  What  is  the  residue  ? 
Test  it. 

If  any  of  the  calculus  still  remains  in  the  test  tube  wash 
it  upon  the  filter  paper  with  some  ether;  wash  the  residue  of 
the  calculus  now  on  the  filter  paper  with  a  10  per  cent  solu- 


BILE.  71 

tion  of  HC1;  then  wash  twice  with  water  and  dry  the  paper 
in  the  funnel. 

When  dry  pass  through  the  filter  again  and  again  about 
10  c.c.  of  chloroform  until  this  assumes  a  yellowish  color, 
caused  by  its  solvent  action  on  the  bilirubin.  Finally  allow 
a  few  c.c.  of  fresh  chloroform  to  flow  through  the  funnel,  and 
uniting  this  with  the  previous  extract,  let  it  evaporate  in  the 
air. 

Treat  the  residue  of  the  calculus  still  on  the  filter-paper 
with  about  10  c.c.  of  alcohol.  What  does  this  extract? 
Evaporate  off  the  alcohol  on  the  water-bath.  Notice  the 
color  of  the  residue  and  compare  it  with  that  of  bilirubin. 

What  was  the  necessity  for  the  use  of  the  hydrochloric 
acid  in  the  analysis? 

Perform  the  following  tests  with  the  residue  of  bilirubin: 

(a)  Gmelin's  test. 

(6)  Dissolve  some  of  the  substance  in  a  few  c.c.  of  chloro- 
form and  shake  it  with  a  dilute  solution  of  Na2C03.  Notice 
the  changes  which  occur  in  the  aqueous  solution. 


BLOOD. 

Blood  may  be  considered  as  composed  of  a  fluid  plasma 
in  which  are  suspended  the  form  elements,  i.e.,  the  red 
corpuscles,  the  leucocytes,  and  the  platelets.  When  the 
plasma  coagulates,  there  separates  from  it  the  insoluble 
fibrin  or  the  clot,  and  the  fluid  which  remains  or  is  pressed 
out  by  the  contraction  of  the  clot,  is  designated  as  the  serum. 
The  reaction  of  the  blood  is  alkaline  to  litmus,  the  alkalinity 
being  equivalent  to  about  0.25  per  cent  Na2C03.  As  was  the 
case  with  the  other  body  fluids,  it  is  also  acid  to  phenol- 
phthalein,  since  it  contains  sodium  bicarbonate  and  dihy- 
drogen  phosphate. 

The  specific  gravity  of  the  blood  varies  within  rather 
small  limits,  1.055-1.066,  and  the  molecular  concentration, 
as  indicated  by  the  depression  of  the  freezing  point,  is  under 
normal  conditions  almost  constant.  Approximately  60  per 
cent  by  weight  of  the  blood  is  composed  of  corpuscles  and 
the  remaining  40  per  cent  of  plasma. 

General  Reactions. 

Test  the  reaction  with  litmus  paper  previously  moistened 
with  a  concentrated  solution  of  NaCl.  To  what  is  this  reac- 
tion due?  Specific  gravity — see  Hammarsten,  p.  223,  Ham- 
merschlag's  method. 

(a)  Examine  a  drop  under  the  microscope. 

(6)  To  5  c.c.  of  blood  add  10  c.c.  of  water.  Notice 
changes  in  the  solution  and  examine  a  drop  under  the  micro- 
scope. What  is  laky  blood? 

(c)  To  5  c.c.  of  blood  add  10  c.c.  of  an  0.8  per  cent  NaCl 
solution.  Examine  a  drop  of  this  also.  What  is  meant  by 

a  solution  isotonic  with  blood? 

73 


BLOOD.  73 

(d)  Add  a  few  drops  of  bile,  chloroform,  and  ether  to 
successive  portions  of  5  c.c.  of  blood. 

(e)  To  5  c.c.  of  blood  add  1  c.c.  of  hydrogen  peroxide. 
To  what  is  the  frothing  due? 

(/)  To  about  10  c.c.  of  water  add  a  few  drops  of  blood 
and  enough  freshly  prepared  tincture  of  guaiacum  to  cause 
a  slight  turbidity.  Then  add  to  the  mixture  a  few  c.c.  of 
hydrogen  peroxide.  Explain  the  effects  produced. 

(g )  Add  10-12  drops  of  a  saturated  glacial  acetic  acid  solu- 
tion of  benzidine  to  2-3  c.c.  of  3%  hydrogen  peroxide.  Mix  and 
to  this  add  2  or  3  drops  of  very  dilute  blood.  Note  change 
in  color.  Try  the  same  reaction  using  dilute  blood  which 
has  previously  been  boiled  and  then  cooled. 

BLOOD  SERUM. 

Blood  serum  presents  a  clear  yellow  liquid  with  a  specific 
gravity  of  1.027-1.032.  It  consists  of  a  watery  solution  of 
serum  albumins,  globulins,  enzymes,  dextrose,  fats,  fibrin 
ferment,  inorganic  salts,  and  a  yellow  coloring  matter  belong- 
ing to  the  class  of  luteins  or  lipochromes.  In  addition  it 
holds  in  solution  traces  of  nearly  every  soluble  substance 
which  has  been  found  in  the  body  tissues.  Of  the  8-9  per 
cent  of  total  solids  in  the  serum,  7  per  cent  is  made  up  of  the 
proteins.  NaCl  is  present  to  the  extent  of  about  65  per 
cent  of  the  inorganic  salts. 

PROTEINS. 

(a)  Heat  about  25-30  c.c.  of  serum  to  boiling  with  the 
addition  of  a  drop  or  two  of  acetic  acid.  Filter  and  test 
filtrate  and  precipitate  for  protein  (Millon's  and  biuret 
reactions).  Retain  the  filtrate. 

(6)  Saturate  15  c.c.  of  the  serum  with  MgS04.  Remove 
the  precipitate  by  filtration,  and  to  the  filtrate  add  two 
drops  of  acetic  acid.  The  filtrate  from  this  second  pre- 


74  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

cipitate  should  be  protein-free.  Test  it.  What  is  the  dif- 
ference between  the  two  precipitates?  What  is  another 
method  for  the  separation  of  albumins  and  globulins? 

(c)  To  5  c.c.  of  the  serum  add  10  c.c.  of  95  per  cent  alcohol. 

(d)  "   5  "    "     "      "        at  30°  C  add  anhydrous  Na2S04 
to  saturation. 

(e)  To  5  c.c.  of  the  serum  add  a  solution  of  uranium 
acetate  until  no  further  precipitate  is  obtained. 

After  the  removal  of  the  precipitates  in  experiments 
(c),  (d),  and  (e),  test  the  nitrates  for  protein. 

SUGAR  AND  SODIUM  CHLORIDE. 

Use  the  protein-free  filtrate  from  experiment  a  (under 
Proteins). 

(a)  Test  a  few  c.c.  of  the  filtrate  with  Fehling's  solution. 

(6)  Allow  a  few  c.c.  to  evaporate  on  a  watch-glass  over 
the  water-bath.  Examine  the  glass  under  the  microscope. 

(c)  Test  for  chlorides. 

BLOOD  PLASMA. 

(a)  To  a  few  c.c.  of  oxalated  plasma  add  a  few  drops  of 
a  2  per  cent  CaCl2  solution.  What  is  the  result?  Why? 

(6)  Dilute  2  c.c.  of  salted  plasma  with  10  volumes  of 
water.  What  happens? 

(c)  Do  the  same  to  some  serum.     What  is  the  difference, 
and  why?. 

(d)  To  5  c.c.  of  the  salt-plasma  add  an  equal  volume  of 
a  saturated  NaCl  solution.     What  is  the  precipitate? 

FIBRIN. 

This  substance  is  formed  by  the  action  of  the  fibrin  fer- 
ment upon  the  globulin,  fibrinogen,  which  is  present  in  all 
the  coagulable  fluids  of  the  body. 


BLOOD.  75 

Note  the  character  of  the  substance  presented.  It  has 
been  obtained  by  whipping  the  blood  before  it  has  had  time 
to  set  in  a  solid  mass. 

Apply  three  color  protein  tests. 

Recall  the  action  of  0.2  per  cent  HC1,  pepsin,  and  trypsin 
upon  fibrin. 

FORM  ELEMENTS. 

These  consist  of  the  red  blood  cells,  the  leucocytes,  and 
the  platelets.  Chemically  the  solid  matter  of  the  red  corpus- 
cles consists  of  90  per  cent  hemoglobin,  8  per  cent  proteins 
and  nucleins,  and  the  remainder  cholesterol,  lecithin,  and 
inorganic  salts.  The  predominating  base  of  these  salts  is 
potassium. 

The  leucocytes,  being  typical  animal  cells,  contain  those 
substances  which  are  intimately  connected  with  the  term 
protoplasm.  Such  are  proteins  (especially  nucleoproteins), 
lecithins,  cholesterols,  fats,  carbohydrates,  inorganic  salts, 
and  water.  Little  is  known  concerning  the  composition  of 
the  blood-platelets.  They  probably  contain  a  protein  of  the 
globulin  type  and  a  nucleoprotein. 

OXYHJEMOGLOBIN. 

Oxyhaemoglobin  or  haemoglobin  belongs  to  the  class  of 
conjugate  proteins  often  spoken  of  as  the  respiratory  pig- 
ments, and  forms  the  most  important  constituent  of  the 
form  elements.  When  its  watery  solution  is  heated  to  70°  C 
it  decomposes  with  the  formation  of  an  iron-containing  pig- 
ment, hcematin,  and  an  albuminous  body,  globin. 

The  function'  of  the  oxyhremoglobin  is  that  of  an  oxygen 
carrier.  The  compound  holds  the  oxygen  in  a  rather  loose 
combination,  and  in  all  probability  the  iron  of  the  hsematin 
molecule  is  directly  concerned  in  the  process.  This  instability 


76  LABORATORY  WORK  IX  PHYSIOLOGICAL  CHEMISTRY. 

of  the  oxygen  combination  renders  the  compound  particularly 
liable  to  the  action  of  reducing  agents.  A  reduction  of  this 
kind  takes  place  in  the  capillaries  of  the  tissues,  hemoglobin 
or  reduced  hemoglobin  being  formed,  the  presence  of  which 
in  predominating  quantities  gives  to  the  venous  blood  its 
characteristic  dark  red  color. 

Hemoglobin  is  very  prone  to  combine  to  form  derivatives 
with  certain  compounds  such  as  carbon  monoxide,  nitric 
oxide,  and  hydrogen  sulphide.  These  simple  substances, 
having  a  greater  affinity  for  the  hemoglobin  molecule  than 
the  oxygen  possesses,  replace  the  latter  readily  and  form 
stable  and  more  or  less  toxic  compounds  which  cause  death 
by  oxygen  starvation.  Again,  partial  decomposition  prod- 
ucts of  hemoglobin  showing  characteristic  spectra  are  easily 
formed  under  certain  conditions;  such  are  methemoglobin, 
hemochromogen,  hematoporphyrin,  etc.  Some  slight  phys- 
ical and  chemical  differences  seem  to  exist  between  the  oxy- 
hemoglobins  obtained  from  the  blood  of  various  animals,  a 
fact  which  explains  why  the  coloring  matter  from  the  blood 
of  different  animals  does  not  crystallize  in  the  same  form  or 
with  the  same  facility. 

Place  on  a  glass  slide  one  drop  of  defibrinated  dog's  blood. 
To  this  add  one  drop  of  water  and  mix  with  a  platinum  wire. 
Allow  the  mixture  to  evaporate  at  room  temperature  until  the 
edges  of  the  drop  have  begun  to  dry.  Then  place  a  cover- 
glass  on  the  slide  and  examine  under  the  microscope.  Sketch 
the  crystals  of  oxyhemoglobin. 

The  proof  for  the  presence  of  oxyhemoglobin  is  usually 
adduced  by  showing  under  the  microscope  crystals  of  hemin. 

Hcemin — Teichmann's  Crystals. 
Hemin  is  the  HC1  ester  of  the  anhydride  of  hematin. 
Place  one  drop  of  NaCl  solution  upon  a  microscopic  slide 


BLOOD.  77 

and  allow  it  to  evaporate  to  dryness.  Then  add  a  very  small 
drop  of  blood  and  two  drops  of  glacial  acetic  acid  and  cover 
with  a  glass.  Warm  cautiously  until  bubbles  of  gas  begin  to 
form  in  the  mixture  under  the  cover-glass.  Examine  and 
sketch  under  the  microscope.  The  hsemin  crystals  are  rhom- 
bic plates,  brown  in  color  by  transmitted  light.  In  large 
masses  they  have  a  metallic  lustre  and  appear  steel-blue  by 
reflected  light. 

Spectroscopic    Examination. 

(a)  Oxyhcemoglobin. — Dilute  1  c.c.  of  blood  with  200  c.c. 
of  water.  Examine  spectroscopically.  At  this  dilution  one 
broad  absorption-band  is  seen  extending  from  the  D  line 
(588)  to  b  (518).  The  violet  end  of  the  spectrum  is  also 
absorbed  as  far  as  the  F  line  (486).  Upon  again  diluting 
this  solution  with  an  equal  volume  of  water  it  is  noticed  that 
the  broad  band  has  resolved  itself  into  two,  the  one  next  to  D 
being  narrower  and  more  intense  than  the  broader  one  to  the 
right.  Between  the  two  bands  is  a  green  interspace.  Less 
of  the  violet  end  is  now  absorbed.  Upon  still  further  dilu- 
tion, the  bands  become  narrower  and  finally  disappear  simul- 
taneously. 

(6)  Hcemoglobin  (Reduced  Hcemoglobiri). — Prepare  some 
Stokes'  reagent  as  follows:  Dissolve  3  grams  of  ferrous  sul- 
phate in  a  small  quantity  of  water  and  add  to  it  in  watery 
solution  2  grams  of  tartaric  acid.  Make  up  the  mixture  to 
100  c.c.  and  just  before  using  add  NH4OH  until  the  precipi- 
tate which  at  first  forms  is  dissolved.  This  solution  of 
ammonium  ferrotartrate  is  a  reducing  agent,  removing  the 
oxygen  which  is  in  weak  combination  with  the  oxyhaBmoglo- 
bin,  and  thus  forming  hemoglobin.  To  the  blood,  200  times 
diluted,  add  a  few  drops  of  Stokes'  reagent.  Notice  the 
change  in  color.  Examine  in  the  spectroscope.  A  broad, 


78  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

less  sharply  defined  absorption-band  is  seen  occupying  as 
much  space  on  the  spectrum  as  the  two  bands  of  oxyhsemo- 
globin,  but  the  haemoglobin  band  is  moved  further  to  the  left. 
If  this  solution  is  shaken  in  the  air  the  color  returns  to  that 
of  oxyhsemoglobin  and  the  latter's  characteristic  spectrum 
also  reappears. 

(c)  Methcemoglobin. — Add  to  blood  (diluted   1:15)  two 
drops  of  a  freshly  prepared  solution  (10%)  of  potassium 
ferricyanide.     The  color  of  the  blood  becomes  brown.     The 
spectrum,  in  addition  to  two  bands  corresponding  nearly  to 
those  of  oxy haemoglobin,  which,  however,  are  only  faintly 
seen,  shows  a  band  in  the  red  near  C.    If  to  such  a  solution, 
while  still  in  position  before  the  spectroscope,  a  drop  or  two 
of  Stokes'  reagent  is  added,  the  characteristic  absorption- 
bands  of  oxyhasmoglobin  appear  for  a  second  and  are  then 
quickly  replaced  by  those  of  haemoglobin.     Shaking  in  the 
air  causes  the  latter  to  be  reoxidized  to  oxyhsemoglobin  with 
the  consequent  spectral  change. 

(d)  Hcemochromogen     (Reduced   Hcematiti).  —  To    blood 
(diluted  1 : 15)  add  two  or  three  drops  of  strong  NaOH  and 
warm  gently  until  the  color  changes  to  a  brownish  green. 
Then  cool  and  add  two  drops  of  Stokes'  reagent.     Such  a  solu- 
tion shows  in  the  spectrum  two  very  dark  bands  coinciding 
apparently   with   those   of   oxyhaemoglobin ;    upon   careful 
examination,  however,  it  will  be  seen  that  green  light  appears 
on  the  left  of  the  left  band  and  consequently  both  bands  must 
be  moved  further  to  the  right  than  the  oxyhsemoglobin  ones. 

(e)  Hcematoporphyrin  (Iron-free  Hcematiri). — Place  a  few 
c.c.  of  concentrated  H2S04  in  a  test-tube  and  add  a  drop  of 
blood,  mixing  well.    The  color  changes  to  wine-red,  and  spec- 
troscopically  the  solution  shows  two  bands  on  the  opposite 
side  of  the  D  line.     The  one  to  the  left  is   narrower  and 
weaker,  while    that   to  the  right  is  much  more   intense, 


BLOOD.  79 

broader,  and  more  sharply  defined.  This  spectrum  is  very 
characteristic. 

(/)  Carbon  Monoxide  Hcemoglobin. — Carbon  monoxide 
haemoglobin  may  be  easily  prepared  by  passing  ordinary  coal- 
gas  through  defibrinated  blood  until  the  latter  assumes  a 
carmine  or  cherry-red  color  characteristic  of  the  combination 
of  CO  with  haemoglobin.  Examined  spectroscopically  such  a 
solution  shows  two  bands  similar  to  oxyhaemoglobin  except 
that  the  bands  of  the  C0-ha3moglobin  spectrum  are  nearer  to 
the  violet  end.  Add  Stokes'  reagent  to  the  solution;  the 
spectrum  remains  unchanged. 

Make  the  following  tests,  first  with  diluted  blood  (oxy- 
haemoglobin)  and  then  with  diluted  CO-hsemoglobin;  note  the 
differences  carefully: 

(a)  Add  to  5  c.c.  of  the  blood  3  c.c.  (NH4)2S. 

(6)     «     «5«     «    „      «     10  c.c.  of  Stokes' reagent. 

(c)  Shake  with  air. 

What  is  characteristic  and  important  in  the  combination  of 
CO  with  haemoglobin  as  shown  by  the  above  experiments? 
Make  drawings  of  all  the  spectra  seen  and  compare  them  with. 
those  in  the  text-books. 


MILK. 

Milk  physiologically  considered  stands  as  a  secretion 
in  the  strictest  sense  of  the  word,  since  its  organic  constitu- 
ents are  specific  products  of  the  activity  of  the  cells  of  the 
mammary  gland.  Chemically  it  presents  a  perfect  emul- 
sion of  fat  whose  menstruum  holds  in  solution  three  proteins 
(lactalbumin,  lactglobulin,  and  caseinogen),  lactose,  inor- 
ganic salts  (chiefly  Ca),  gases,  and  traces  of  creatinine,  leci- 
thin, cholesterol,  urea,  and  citric  acid.  In  the  comparison 
of  the  composition  of  human  and  cow's  milk,  the  chief  point 
to  be  noted  is  the  relatively  low  percentage  of  total  solids, 
protein  and  fat,  and  high  percentage  of  lactose  in  human 
as  against  cow's  milk.  Fresh  milk  possesses  either  an  ampho- 
teric  or  alkaline  reaction,  but  with  phenolphthalein  it  also 
has  a  certain  acidity.  Upon  standing,  it  develops  a  definite 
acid  reaction  by  the  change  of  the  lactose  into  lactic  acid. 
This  throws  the  caseinogen  out  of  solution  and  the  milk  is 
said  to  be  sour.  Fresh  milk  does  not  coagulate  by  heat,  but 
forms  a  scum  supposed  to  consist  of  some  form  of  caseinogen 
and  calcium  salts. 

The  specific  gravity  of  human  and  cow's  milk  is  approxi- 
mately the  same  (1.028-1.034).  It  is  increased  by  the  re- 
moval of  the  fat  (cream),  which  has  a  specific  gravity  lower 
than  that  of  water. 

General  Reactions. 

(a)  Examine  under  the  microscope  fresh  milk,  skimmed 
milk,  and  colostrum.  What  are  the  differences? 

80 


MILK.  81 

(6)  Take  the  specific  gravities  and  explain  the  variations 
in  the  results. 

(c)  Test  the  reaction  with  litmus  paper. 

(d)  Saturate  10  c.c.  of  milk  with  MgSO4.    What  is  the 
precipitate? 

(e)  Shake  5  c.c.  of  milk  with  ether.    What  takes  place? 
Now  add  a  few  drops  of  NaOH.     Explain  this  change. 

(/)  Heat  some  milk  in  an  evaporating-dish.  Notice  the 
scum. 

(</)  Heat  5  c.c.  of  milk  and  5  c.c.  of  NaOH  to  boiling. 
The  yellowish-brown  coloration  is  caused  by  the  action  of 
the  alkali  on  the  lactose. 

(/i)  Try  the  guaiac  reaction  described  under  blood,  using 
milk  instead.  Explain  the  result. 

QUALITATIVE  SEPARATION  OF  THE  CONSTITUENTS 
OF  MILK. 

Dilute  200  c.c.  of  milk  with  800  c.c.  of  water  and  add  weak 
acetic  acid,  drop  by  drop,  until  a  white  flocky  precipitate 
separates  out,  leaving  a  fairly  clear  solution.  Allow  the  pre- 
cipitate to  settle;  then  filter  (Filtrate  A)  it  off  and  let  it 
drain  as  dry  as  possible.  Remove  the  white  precipitated 
caseinogen  from  the  paper  and  place  it  in  a  flask  with  200  c.c. 
of  a  mixture  one-half  ether  and  one-half  alcohol.  Allow  the 
precipitate  to  stand  in  contact  with  the  ether  for  an  hour, 
shaking  from  time  to  time.  Then  filter  off  the  ether  mix- 
ture and  let  it  evaporate  spontaneously.  The  residue  con- 
sists of  most  of  the  fat  of  the  milk,  mechanically  precipitated 
adherent  to  the  caseinogen.  Keep  both  the  caseinogen  and 
the  fatty  residue. 

Place  the  filtrate  A  in  an  evaporating-dish  and  heat  over 
a  free  flame ;  the  solution  being  acid,  any  coagulable  protein 


82  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

not  precipitated  with  the  acetic  acid  will  be  rendered  insolu- 
ble. When  the  solution  has  concentrated  to  about  one- 
half  its  original  volume,  this  may  be  filtered  off  and  the  clear 
filtrate  again  set  evaporating  until  crystals  begin  to  appear, 
separating  out  of  the  solution  (now  about  20  c.c.).  Keep 
the  coagulable  protein  precipitate. 

After  allowing  the  solution  to  cool,  filter  off  the  crystals  of 
calcium  phosphate  and  again  place  the  filtrate  over  the  water- 
bath  and  allow  it  to  evaporate  to  a  thick  syrup.  Of  what  is 
this  composed? 

From  the  milk  have  now  been  isolated— 

1.  Caseinogen.  2.  Fats.  3.  Coagulable  protein.  4.  Cal- 
cium phosphate.  5.  Lactose. 

These  require  separate  study. 

CASEINOGEN. 

This  substance  is  a  non-coagulable  protein  of  the  class  of 
phosphoproteins.  It  contains  about  0.8%  phosphorus  and 
0.7-1.2%  sulphur.  Caseinogen  deports  itself  chemically 
as  a  weak  acid  combining  with  bases  to  form  soluble  salts 
or  caseinogenates.  The  acid  itself  is  insoluble.  It  exists 
in  the  milk  in  the  form  of  Ca-caseinogenate  (apparently  not 
in  a  true  solution,  however),  and  when  stronger  acids  are 
added  caseinogen  is  formed  and  falls  out  of  solution. 

(a)  Try  three  color-protein  tests. 

(6)  Test  for  phosphorus  and  sulphur  by  fusion. 

(c)  Dissolve  some  in  very  dilute  NH4OH  and  reprecipitate 
with  acetic  acid. 

(d)  Test  for  lead-blackening  sulphur. 

(e)  Grind  up  a  little  of  the  substance  in  a  mortar  with 
CaC03  and  water,  then  filter  and  in  the  filtrate  test  for  case- 
inogen.   What  chemical  reaction  has  taken  place  here? 


MILK.  83 

FATS. 

The  fats  of  milk  are  in  general  those  which  occur  in 
adipose  tissue.  There  are  present  also  in  small  quantities 
fats  formed  from  the  volatile  fatty  acids,  particularly  butyric 
and  caproic  acids.  These  give  to  milk-fat  (butter)  its  charac- 
teristic odor  when  from  any  cause  the  fatty  acids  are  liberated 
(rancidity).  The  proportion  of  the  various  fats  is  roughly 
as  follows:  triolein,  -|;  tripalmitin,  ^ ;  tristearin,  £;  tributyrin 
and  tricaproin,  ^. 

Use  the  fatty  residue  in  the  evaporating-dish  (see  method 
of  separation).  Dissolve  the  fat  in  10  c.c.  of  ether  and  add 
to  it  3-5  c.c.  of  sodium  alcoholate.  Notice  what  takes  place 
and  compare  this  saponification  with  those  performed  under 
fats.  Evaporate  off  the  ether  and  replace  it  with  water. 
The  precipitate  should  dissolve.  Now  add  dilute  H2S04  to 
a  distinct  acid  reaction. 

Note  the  odor.  To  what  is  it  due?  Write  the  equations 
for  the  reactions. 

COAGULABLE  PROTEIN. 

Lactalbumin  and  lactglobulin  together  occur  to  the 
extent  of  1  per  cent  in  cow's  milk.  They  are  closely  related 
to,  though  not  identical  with,  serum  albumin  and  serum 
globulin. 

Try  three  color-protein  tests. 

CALCIUM  PHOSPHATE. 

In  the  inorganic  salts,  phosphoric  forms  the  chief  acid 
and  calcium  the  base,  except  in  human  milk,  where  potas- 
sium seems  to  predominate.  Strangely  enough,  iron  is  only 
present  in  traces. 

Dissolve  some  in  dilute  HN03. 


84  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY 

(a)  Add  an  equal  volume  of  ammonium  molybdate  and 
warm  to  70°  C.  What  is  the  precipitate?  Filter  it  off,  dis- 
solve in  NH4OH  and  reprecipitate  as  ammonio-magnesium 
phosphate. 

(6)  Make  a  portion  of  the  HN03  solution  alkaline  with 
NH4OH  and  reacidify  with  acetic  acid.  Add  a  few  c.c.  of 
ammonium  oxalate.  Examine  the  precipitate  microscopic- 
ally. 

LACTOSE. 

Lactose  occurs  only  in  milk,  if  the  small  amounts  which 
are  occasionally  present  in  the  blood  and  urine  of  pregnant 
women  are  disregarded.  It  is  a  disaccharide  and  breaks 
down  into  a  molecule  of  dextrose  and  one  of  galactose.  It 
undergoes  yeast  fermentation  very  slowly,  but  is  easily 
attacked  by  bacterium  lactis,  in  which  process  lactic  acid  is 
formed.  With  phenylhydrazin,  lactose  yields  osazones,  which 
crystallize  in  needles  and  melt  at  200°  C.  Lactose  is  soluble 
in  water,  but  does  not  dissolve  in  either  alcohol  or  ether. 
Its  "solutions  are  dextrogyrate. 

Make  use  of  the  syrup  derived  from  the  last  evaporation 
in  the  milk-separation. 

(a)  Try  Fehling's,  Moore's,  and  Ny lander's  tests. 

(6)  Apply  the  fermentation  test. 

(c)  Try  the  phenylhydrazin  test  (see  p.  5). 

(d)  To  5  c.c.  of  the  lactose  solution  add  sufficient  freshly 
prepared  indigo-carmine  solution    to    render    the    mixture 
decidedly  blue;    then  make  it  alkaline  with  dilute    Na2C03 
solution.     Upon  warming,  the  mixture  becomes  red,  yellow, 
and  finally  colorless.    What  do  these  changes  indicate? 


URINE. 

Considered  from  the  physiological  point  of  view,  the 
urine  forms  the  chief  exit  for  those  soluble  substances  which, 
either  on  account  of  their  toxicity  under  certain  conditions 
or  from  their  lack  of  available  potential  energy,  are  unsuited 
for  utilization  and  further  retention  in  the  organism.  Chem- 
ically, the  urine  presents  a  watery  solution  of  organic  and 
inorganic  bases  and  acids,  whose  degree  and  manner  of  com- 
bination is  dependent  upon  the  conditions  (affinity,  mass- 
action,  etc.)  existent  in  the  solution.  The  compounds 
present  in  the  urine  represent,  on  the  one  hand,  the  end 
products  of  combustion  and  cellular  metabolism,  and,  on 
the  other,  bodies  excreted  in  the  same  or  similar  form  in 
which  they  were  ingested.  It  is  this  double  origin  of  the 
constituents  of  the  urine  which  necessitates  a  comprehensive 
knowledge  of  the  daily  ingesta,  if  rational  or  accurate  de- 
ductions are  to  be  made  from  the  composition  of  the  urine. 
Again,  as  the  character  and  composition  of  the  urine  ex- 
creted at  different  periods  of  the  day  varies  within  rather 
wide  limits,  it  is  absolutely  essential  in  every  examination 
of  urine  that  the  sample  presented  for  analysis  should  repre- 
sent a  definite  part,  if  not  the  whole,  of  a  well-mixed  24-hours 
excretion.  This  in  part  affords  an  explanation  of  the  now 
current  method  of  considering  the  constituents  of  the  urine 
from  the  standpoint  of  weight  per  diem  (grammes)  rather  than 
concentration  (per  cent).  Percentage  data  neglect  one 

85 


86  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

important  factor  (volume),  the  consideration  of  which  is 
indispensable,  especially  when  extreme  values  are  met  with. 
It  is  unnecessary  to  add  that  the  urine  should  be  ex- 
amined as  fresh  as  possible,  or,  in  the  event  of  this  not  being 
feasible,  the  fluid  must  be  well  supplied  with  antiseptic  agents 
such  as  powdered  thymol,  chloroform,  NaF,  etc. 

Physical  Properties. 

The  following  comprise  the  important  physical  properties 
of  urine  to  be  specially  noted  in  an  examination: 

Color. — Aromatic  alcohols,  salol  and  allied  compounds 
cause  the  urine  to  become  brown  upon  standing.  Rhubarb 
and  senna  impart  a  reddish  color,  which,  if  the  urine  is 
alkaline,  may  be  confounded  with  the  appearance  of  blood. 
The  ingestion  of  quinine  in  large  quantities  gives  to  the 
urine  a  greenish  opalescence  and  methylene  blue  is  followed 
by  the  appearance  of  urine  of  a  decided  bluish-green  tinge. 

It  is  usual  to  refer  to  Vogel's  color  chart,  but  unless  the 
color  is  other  than  that  of  some  shade  of  yellow,  little  can  be 
deduced  from  the  determination  of  this  property. 

Odor. — Normal  (urinous),  ammoniacal,  or  that  peculiar  to 
some  substance  not  normally  present,  such  as  acetone, 
methyl  mercaptan,  etc. 

Transparency. — Clear  or  cloudy.  Usually  upon  standing, 
a  cloud  of  mucous-like  substance  separates,  suspending  itself 
in  some  part 'of  the  urine. 

Reaction. — Acid,  neutral,  or  alkaline. 

As  is  true  of  any  acid  solution,  the  acidity  of  the  urine 
is  due  to  the  presence  of  dissociated  hydrogen  atoms  (ions). 
It  is  usually  assumed  that  these  acid  ions  are  derived  from 
the  acid  salts,  such  as  H2NaP04  or  HNa-jPC^  (mineral  acidity), 
but  in  all  probability  the  H  ions  of  the  organic  acids  in  the 
urine  also  add  a  not  inconsiderable  part  to  the.  total  acidity. 


URINE.  87 

Free  mineral  acids  are  never  present.  By  a  proper  relationship 
between  the  dihydrogen  and  monohydrogen  phosphates,  a 
neutral  or  amphoteric  reaction  may  prevail  (litmus).  Under 
normal  conditions  the  24-hour  urine  is  never  alkaline;  but 
portions  of  urine  drawn  a  few  hours  after  digestion  may 
react  alkaline,  due  to  the  withdrawal  of  acidic  radicals 
(acid  ions)  from  the  blood  in  the  formation  of  the  acid  gastric 
juice.  Alkaline  urine  may  be  caused  by  free  or  fixed  alkalies 
and  when  this  is  the  case  the  urine  is  always  cloudy  or  turbid. 
Care  should  be  taken  that  the  observed  alkalinity  is  not  a 
result  of  bacterial  contamination.  Volatile  alkalies  such 
as  ammonia  react  blue  to  litmus,  but  upon  warming  the 
paper  the  red  color  returns ;  this  will  not  occur  if  the  reaction 
is  caused  by  a  fixed  alkali.  Organic  acids  are  burnt  in 
the  body  to  carbonates  and  the  latter  decrease  the  acidity, 
sometimes,  in  fact,  causing  an  alkaline  reaction  to  appear. 

Volume  and  Specific  Gravity. — Under  normal  conditions 
these  two  factors  vary  inversely  with  each  other;  a  large 
volume  of  urine  is  usually  accompanied  by  a  low  specific 
gravity  and  vice  versa.  The  volume  depends  upon  the 
amount  of  water  ingested  and  that  excreted  by  the  bowels, 
skin,  and  lungs;  it  varies  between  800  and  1500  c.c.  (aver- 
age 1250  c.c.).  The  specific  gravity  may  fall  between  1.010 
and  1.030  (average  1.017-1.020). 

The  volume  should  be  measured  in  a  graduated  cylinder 
capable  of  holding  two  liters.  Should  it  be  required  to 
retain  the  urine  for  some  time,  thymol  must  be  added  and 
the  fluid  placed  in  a  stoppered  vessel. 

Determination  of  the  specific  gravity  is  made  by  means 
of  the  urmometer  (hydrometer).  If  there  is  a  sediment  the 
urine  must  be  warmed,  filtered,  and  allowed  to  cool. 

Place  the  cylinder  upon  the  table  and  fill  it  to  within  an 
inch  of  the  top  with  urine;  then  immerse  the  urinometer. 


88  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

Read  off  on  the  graduation  of  the  spindle  the  mark  where 
the  meniscus  of  the  fluid  cuts  it. 

Sediments. — Notice  should  be  taken  as  to  their  color, 
amount,  character,  etc.  The  method  for  the  determination 
of  the  identity  of  such  will  be  taken  up  later. 

Total  Solids. — These  may  be  calculated  approximately  by 
multiplying  the  second  and  third  decimals  of  the  specific 
gravity  by  Haser's  coefficient  =  2. 33.  This  gives  the  number 
of  grammes  in  1000  c.c.  of  the  urine,  from  which  must  be 
calculated  the  total  amount  in  the  twenty-four  hours. 

THE    NORMAL    CONSTITUENTS    OF    THE    URINE. 
INORGANIC  COMPOUNDS. 

Concerning  the  exact  nature  of  the  combinations  formed 
in  the  urine  by  the  various  bases  and  acids  (ions)  but  little 
is  known  at  present.  The  compounds  are  present  in  rather 
dilute  solution  and  as  such  are  amenable  to  all  the  chemical 
and  physical  laws  of  salts  in  like  solutions,  complicated, 
however,  by  the  fact  that  the  condition  is  not  one  of  a  single 
salt  in  a  simple  solution  but  of  an  exceedingly  complex  mix- 
ture of  salts  both  inorganic  and  organic.  The  dissociation 
of  one  salt  is  influenced  by  the  presence  of  others,  also  disso- 
ciated, and  the  particular  combinations  of  the  various  (ions) 
bases  or  acids  depend  upon  their  relative  masses  and  avidities. 
Again,  as  regards  two  compounds  with  like  ions  each  de- 
creases the  solubility  of  the  other,  while  the  solubility  of 
unlike  ions  is  increased  by  their  mutual  presence. 

These  factors,  dissociation,  mass  action,  and  affinity  deter- 
mine the  character  of  the  ionic  equilibrium  which  is  present 
in  the  urine,  but  as  to  the  exact  nature  of  such  equilibrium, 
our  present  methods  tell  next  to  nothing.  The  later  work 


URINE.  8& 

along  physical  lines  has  been  in  this  direction,  in  the  hope 
that  the  results  will  be  of  value  for  diagnostic  purposes. 

At  present  it  is  customary  to  determine  the  quantity 
of  a  given  base  or  acid  radical  and  then  to  express  it 
empirically  in  terms  of  a  compound  of  this  radical,  the 
quantity  of  which  is  considered  to  predominate  in  the  mix- 
ture. Thus  chlorine  is  expressed  as  NaCl,  potassium  as  K20, 
etc.  The  ordinary  inorganic  acidic  radicals  (anions)  of  the 
urine  are  Cl',  SO/',  PO/",  and  CO/'  and  the  basic  radicals; 
(kations),  NH/,  1C,  Na',  Ca",  Mg",  with  traces  of  Fe". 

ACIDIC  RADICALS. 

(a)  Chlorides. — These  are  present  combined  principally 
with  Na  and  K.  Acidify  some  of  the  urine  with  HN03  and 
add  a  drop  of  AgN03.  Prove  that  this  precipitate  is  AgCl. 

(&)  Sulphates. — These  occur  in  two  forms,  viz.,  preformed 
or  sulphate-sulphuric  acid  and  ethereal  or  combined  sulphuric 
acid.  The  former  precipitates  directly  from  the  urine  by  the 
addition  of  BaCl2  in  an  acid  reaction,  the  latter  only  after  the 
previous  boiling  of  the  urine  with  a  mineral  acid.  Acidify 
some  urine  with  acetic  acid  and  add  an  excess  of  BaCl2. 
Prove  that  this  precipitate  is  BaS04.  Filter  (filtrate  must 
be  clear),  and  to  this  add  a  few  c.c.  of  concentrated  HC1  and 
boil  a  few  minutes.  A  turbidity  is  indicative  of  the  presence 
of  ethereal  sulphates.  Explain  this. 

Sulphur  in  the  organic  or  lead-blackening  form  may  also 
be  present  in  the  urine  as  a  constituent  of  taurine,  cystine, 
KSCN,  etc.  Place  some  urine  in  a  flask  with  a  few  pieces  of 
pure  zinc  and  add  enough  HC1  to  cause  a  gentle  evolution  of 
gas.  Partially  close  the  mouth  with  a  piece  of  filter-paper 
moistened  with  lead  acetate.  In  the  presence  of  organic  sul- 
phur the  paper  is  blackened.  Explain  this. 


90  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

(c)  Phosphates. — These  are  present  combined  with  NH4, 
Na,  K,  Ca,  and  Mg  as  primary,  secondary,  or  tertiary  com- 
pounds. 

1.  Make  some  urine  alkaline  with  NH4OH.     What  is  the 
precipitate?    Filter  and  to  the  clear  filtrate  add  two  drops 
of  BaCl2.   .What  is  this  new  precipitate?    Filter  this  off 
through  the  same  filter  as  the  first  and  dissolve  the  whole 
precipitate  in  dilute  HN03.     Test  for  phosphates. 

2.  Make  the  urine  acid  with  acetic  acid  and  add  a  few 
drops  of  uranium  nitrate  solution.     Of  what  is  the  precipitate 
composed? 

3.  Acidify  the  urine  with  a  few  c.c.  of  HN03  and  add 
some  molybdic  solution.    Warm  at  80°  C.     What  is  this 
precipitate? 

4.  Boil  about  10  c.c.  of  the  urine  and  add  one-quarter  its 
volume  of  Fehling's  solution.     Notice  the  color  of  the  pre- 
cipitate.   To  what  is  it  due?    Then  boil  10  c.c.  of  Fehling's 
solution  and  add  one-quarter  its  volume  of  urine.    What 
occurs?    Contrast  these  two  experiments. 

Write  the  equations  for  the  reactions  in  the  above  experi- 
ments. 


BASIC  RADICALS. 

With  the  exception  of  NH4  these  have  little  or  no  chemi- 
cal significance.  The  proof  for  the  presence  of  ammoniacal 
compounds  will  be  taken  up  under  the  quantitative  deter- 
minations. 


URINE.  91 


ORGANIC  COMPOUNDS. 
NH2 

UREA,  OC< 

XNH2 

Urea  or  carbamide  is  present  in  the  urine  in  about  a 
2  per  cent  solution  or  30  grms.  for  24  hours.  It  is  very 
soluble  in  water  and  alcohol  and  crystallizes  in  long  rhombic 
prisms.  It  possesses  weakly  basic  properties  and  forms  com- 
binations with  acids  analogous  to  double  salts.  When 
heated  gently,  urea  yields  biuret.  The  Micrococcus  urece 
splits  it  into  NH3  and  C02. 

Prepare  some  urea  from  the  urine  as  follows-: 

Evaporate  one-half  a  liter  of  urine  to  syrupy  consistency 
and  exhaust  the  residue  with  hot  alcohol.  Filter  off  the 
latter  and  evaporate  the  nitrate  to  dryness.  This  residue 
is  extracted  on  the  water-bath  with  successive  portions  of 
pure  acetone,  which  must  be  filtered  hot.  The  mixed  acetone 
filtrates  are  then  evaporated  slowly  to  a  small  volume  and 
allowed  to  cool.  The  crystals  which  separate  out  may  be 
filtered  off  and  washed  with  cold  acetone. 

Make  use  of  these  urea  crystals  for  the  following  reac- 
tions : 

(a)  Dissolve  some  urea  in  a  few  c.c.  01  water.  Place  in 
each  of  two  watch-glasses  one  drop  of  the  solution.  To  one 
add  one  drop  of  dilute  HNO3  and  to  the  other  a  drop  of  dilute 
oxalic  acid  solution.  Allow  them  to  stand  and  then  examine 
the  crystals  under  the  microscope.  What  are  these  com- 
pounds? 

(6)  Warm  carefully  a  few  crystals  of  urea  in  a  dry  test- 
tube.  The  substance  melts,  giving  off  NH3.  Continue  heat- 


92  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

ing  until  the  fused  mass  solidifies.  Cool  the  test-tube  and 
add  a  few  c.c.  of  water  and  NaOH  drop  by  drop  until 
the  residue  is  all  in  solution.  Add  now  a  drop  or  two  of 
very  dilute  CuS04  solution.  To  what  reaction  does  this 
correspond?  What  substance  is  formed  from  the  urea? 
Write  the  equation. 

(c)  To  5  c.c.  of  NaOH  add  some  bromine  water  and 
drop  in  a  crystal  of  urea.  What  is  the  reaction  which  takes 
place? 

HN— CO 

I        I        H 
URIC  ACID,    CO  C— N\ 

>CO 


HN— C— X 


II 


Uric  acid  is  a  diureide  and  structurally  is  composed  of 
two  urea  groups  attached  to  a  3-carbon  chain.  It  is  closely 
related  to  the  purine  bases  being  built  up  from  the  "purine" 
type. 

Uric  acid  acts  as  a  weak  dibasic  acid  and  forms  three 
types  of  salts — neutral,  biurate,  and  quadriurate. 

The  neutral  salts  which  are  formed  by  the  replacement 
of  two  hydrogen  atoms  of  the  acid  by  two  atoms  of  the  base 
are  very  unstable. 

The  biurates  have  only  one  of  the  hydrogens  replaced 
and  form  very  stable  compounds.  They  present  the  chief 
type  in  which  uric  acid  exists  in  the  urine. 

The  quadriurates  stand  between  the  other  urates  as  re- 
gards stability.  They  are  formed  by  a  weak  combination 
of  uric  acid  and  biurate  molecule  for  molecule.  They 
therefore  contain  in  the  molecule  one-fourth  the  quantity 
of  base  that  exists  in  the  neutral  urate.  When  uric  acid 
separates  out  spontaneously  from  a  urine,  it  is  caused  by  an 


URINE.  93 

inter-reaction  between  the  acid  phosphates  and  the  biurates 
in  which  quadriurates  are  formed;  the  latter  immediately 
break  up  into  uric  acid  and  biurate,  and  the  former  is 
thrown  out  of  solution. 

Pure  uric  acid  forms  a  white  powder.  As  it  separates 
out  from  urine  in  the  presence  of  impurities,  it  assumes  a  great 
diversity  of  crystalline  forms,  all  of  which  are  characteristic, 
however;  the  crystals  are  always  tinged  with  pigment  de- 
rived from  the  urine. 

Uric  acid  is  practically  insoluble  in  hot  and  cold  water, 
alcohol  or  ether.  The  sodium,  potassium,  and  lithium  salts 
are  soluble,  especially  the  latter.  The  ammonium,  calcium, 
and  magnesium  salts  are  insoluble.  The  quantity  excreted 
in  24  hours  varies  according  to  the  individual  and  the  diet, 
but  usually  amounts  to  0.5 — 1.0  grms.  (average  0.7  grm.). 
If  urine  is  made  sufficiently  acid  with  HC1  to  react  strongly 
to  litmus  (about  30  c.c.  cone.  HC1  to  the  liter)  and  allowed 
to  stand,  uric  acid  will  separate  out  in  crystals  of  a  dark 
red  color. 

For  the  following  tests  make  use  of  the  uric  acid  prepared 
in  this  way: 

(a)  Examine  the  crystals  under  the  microscope.  Sketch 
as  large  a  variety  as  can  be  found. 

(6)  Test  the  solubility  in  water,  NaOH  and  NH4OH. 

(c)  Dissolve  some  of  the  crystals  in  a  few  c.c.  of  dilute 
NaOH  and  then  add  NH4C1  to  saturation.     What  is  the  pre- 
cipitate? 

(d)  Heat  a  crystal  on  platinum-foil. 

(e)  Dissolve  some  uric  acid  in  a  small  quantity  of  dilute 
NaOH.     Add  concentrated  H2S04  carefully,  drop  by  drop, 
until  the  solution  is  too  warm  to  touch;  then  add  a  few  c.c. 
of  potassium  permanganate  solution.     What  is  the  reaction 
which  takes  place? 


94  LABORATORY  WORK  IX  PHYSIOLOGICAL  CHEMISTRY. 

(/)  Make  a  concentrated  solution  of  uric  acid  and  pour  it 
into  10  c.c.  of  Fehling's  solution  which  has  previously  been 
brought  to  boiling.  Observe  and  note  result. 

(g)  Dissolve  some  uric  acid  in  dilute  Na2C03  and  place 
a,  couple  of  drops  on  some  filter-paper  previously  moistened 
with  AgN03.  To  what  is  the  blackening  due? 

(h)  Murexide  Test. — Place  a  few  crystals  in  a  clean  and 
dry  evaporating-dish  and  pour  upon  them  two  drops  of 
concentrated  HN03.  Evaporate  to  dryness  very  carefully 
over  a  free  flame.  A  yellowish  residue  results  which  upon 
cooling  and  the  addition  of  a  drop  of  NH4OH  becomes  purple- 
red.  If  NaOH  instead  be  used,  the  color  will  be  purple- 
violet.  What  is  the  chemistry  of  the  reaction? 

PURINE  BASES. 

These  substances  as  they  appear  in  the  urine  have  a 
double  origin.  They  may  be  the  result  of  ingested  nu- 
cleins  (exogenic  origin)  and  also  the  end  products  of  the 
nuclear  metabolism  of  the  tissues  (endogenic  origin).  Al- 
though ten  different  bases  have  been  isolated,  it  is  question- 
able if  many  of  them  are  not  laboratory  products  formed 
during  the  complicated  processes  of  their  isolation.  Xan- 
thine,  hypoxanthine,  guanine,  adenine,  epiguanine,  para- 
xanthine,  and  carnine  are  said  to  be  present  in  the  urine. 
Taken  together  the  quantity  daily  excreted  is  only  about 
20-100  mg.  or  about  10  per  cent  of  the  uric  acid.  They  are 
capable  of  forming  insoluble  compounds  with  Ag,  Cu,  phos- 
photungstic  acid,  etc. 

To  20  c.c.  of  urine  add  an  excess  of  the  magnesium  mix- 
ture. Filter,  and  to  the  filtrate  add  ammoniacal  silver 
nitrate  solution.  The  precipitate  is  composed  of  the  Ag 
compounds  of  all  the  bases.  This  is  filtered  off,  suspended 
in  water,  decomposed  with  H2S,  and  the  Ag2S  removed  by 
filtration.  The  clear  filtrate  is  evaporated  to  dryness.  This 


URINE  95 

residue  is  treated  with  3%  H2S04  which  dissolves  the  purine 
bases  and  leaves  the  uric  acid  undissolved.  The  dissolved 
purine  bases  may  be  reprecipitated  with  silver  nitrate  solution. 

XNH CO 

CREATININE,  HN=C\ 

XN-CH3-CH2 

Creatinine  appears  in  the  urine  as  the  result  of  the  inges- 
tion  of  meat  which  contains  creatine.  The  amount  is  also 
somewhat  dependent  upon  the  nitrogenous  metabolism, 
being  decreased  in  starvation.  For  the  relationship  be- 
tween creatine  and  creatinine  and  the  reactions  and  tests, 
see  under  Muscle.  On  a  mixed  diet  about  0.1  grm.  is  ex- 
creted in  24  hours. 

Creatinine  allows  of  a  separation  from  the  urine  as 
follows : 

To  50  c.c.  of  urine  add  3  c.c.  of  a  saturated  solution  of 
sodium  acetate  and  then  10  c.c.  of  a  saturated  solution  of 
HgCl2.  Filter  off  the  precipitated  urates,  sulphates,  and 
phosphates,  and  set  the  nitrate  aside  for  24  hours.  The 
mercury  compound  of  creatinine  separates  out  in  the  form 
of  spherical  globules.  Examine  some  under  the  microscope. 
This  compound  of  creatinine  is  readily  decomposed  by  acids 
or  by  H2S.  Try  Weyl's  or  Jaffe's  test  directly  on  the  urine 
and  then  on  the  creatinine  isolated  by  the  above  method. 
Test  its  reducing  power. 

CONJUGATE  SULPHATES. 

Of  the  toxic  substances  which  are  formed  as  the  result  of 
intestinal  putrefaction  of  the  proteins,  phenol,  p-cresol, 
indole,  and  skatole  appear  in  the  urine  in  the  form  of  alkali 
salts  of  non-toxic  ethereal  combinations  with  sulphuric 
acid;  pyrocatechinol  and  hydrochinol  are  also  excreted  in 
the  same  way.  Taken  together  these  compounds  are  denoted 


96  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

as  the  ethereal  or  conjugated  sulphates.  The  strength  of 
combination  and  manner  of  detection  of  the  sulphate  radical 
has  been  studied  under  the  inorganic  salts.  The  amount 
of  the  conjugate  sulphates  varies  from  0.09  to  0.6  grm., 
being  dependent,  as  it  would  be  expected  from  their  origin, 
upon  the  extent  of  putrefaction  in  the  intestine. 

The  Phenol  and  p-Cresol  sulphates  exist  in  normal 
urine  in  relatively  small  amounts,  about  30  mg.  for  twenty- 
four  hours.  They  may  be  isolated  and  detected  as  follows: 

Treat  the  urine  with  |  its  volume  of  25  per  cent  H2S04 
and  distil  off  TV  of  the  volume  of  the  solution.  Use  this 
distillate  for  the  succeeding  reactions: 

(a)  Add  some  Millon's  reagent  to  a  little  of  the  solution. 
Warm.  Compare  this  with  the  test  under  protein  and 
tyrosine. 

(6)  To  a  few  c.c.  of  the  solution  add  a  trace  of  neutral 
ferric  chloride  solution.  When  did  you  use  this  reaction 
before?  Add  a  drop  of  HC1. 

(c)  To  10  c.c.  of  the  distillate  add  some  bromine-water. 
Note  the  whitish  crystalline  precipitate  of  tribromphenol. 

The  combinations  with  Pyrocatechinol  and  hyurochinol 
originate  after  the  ingestion  of  these  bodies  or  of  phenol. 
The  urine  containing  them  becomes  brown  on  standing  ("car- 
bolic urines  "). 

Indole  and  Skatole  (/3-methyl  indole)  do  not  unite  with 
the  sulphuric  acid  directly  as  such,  but  first  suffer  an  oxida- 
tion by  which  indoxyl  and  skatoxyl  are  formed;  they  then 
combine  and  appear  in  the  urine  as  potassium  indoxyl  or 
skatoxyl  sulphate.  The  former  is  called  animal  indican 
and  is  not  to  be  confounded  with  the  indican  of  plants.  The 
skatoxyl  compound  is  only  present  in  minimal  quantities 
and  not  infrequently  is  absent  entirely. 


URINE.  07 


INDICAN= POTASSIUM  INDOXYL  SULPHATE, 
C.H/NH^H 


The  following  tests  are  based  upon  the  oxidation  of  the 
indican  to  indigo,  and  the  solvent  action  of  chloroform  upon 
the  latter. 

(a)  Jaffe's  Test. — To  about  10  c.c.  of  urine  add  2  or  3  c.c. 
of  chloroform  and  mix  well  with  an  equal  volume,  10  c.c.,  of 
concentrated  HC1.  Then  add  drop  by  drop,  shaking  well 
between  each  drop,  a  concentrated  solution  of  chloride  of 
lime.  The  indigo  is  dissolved  by  the  chloroform.  Note  the 
color  changes. 

(6)  Obermayer's  Test. — Perform  the  test  in  the  same  man- 
ner except  instead  of  concentrated  HC1  and  lime  add  an  equal 
volume  of  Obermayer's  reagent. 

(c)  Hammarsteris  Test. — The  same  test,  using  Hammar- 
sten's  reagent.  Compare  results. 


c 


OXALIC  ACID, 

\OH 


Oxalic  acid  is  present  in  the  normal  urine  in  the  form  of 
calcium  oxalate,  which  is  held  in  solution  by  the  presence  of 
the  acid  phosphates.  Frequently  the  salt  separates  out  in 
crystalline  form  (see  sediments)  and  then  lays  the  foundation 
for  renal  and  vesical  calculi.  The  quantity  averages  about 
30  mg.  for  24  hours.  In  certain  conditions  which  are  not 
well  understood  the  amount  is  largely  increased  (oxaluria). 
The  oxalic  acid  originates  in  part  from  the  oxalates  ingested 


98  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

in  vegetable  food,  but  as  it  does  not  disappear  in  starvation, 
some  must  arise  from  a  particular  phase  of  protein  metabolism, 
possibly  the  nucleins. 

The  following  is  the  procedure  employed  for  its  isolation: 
The  urine  is  acidified  (20  c.c.  of  HC1  sp.  gr.  1.12  for  every 
1000  c.c.)  and  extracted  with  a  10  per  cent  solution  of  alcohol 
in  ether.  Three  extractions  are  united,  evaporated  to  10-20 
c.c.,  filtered  and  the  filtrate  made  slightly  alkaline  with 
NH4OH;  to  this  is  added  a  few  c.c.  of  1  per  cent  CaCl2  solu- 
tion and  the  mixture  is  rendered  faintly  acid  with  acetic 
acid.  Collect  the  precipitate  on  a  small  filter  and  examine 
it  under  the  microscope.  Heat  some  on  a  platinum  foil. 

HIPPURIC  ACID,  C6H6-CO-NH-CH2-C/QH 

This  substance  is  formed  in  the  renal  cells  by  a  syn- 
thesis of  benzoic  and  aminoaceticacid  or  glycocoll.  Normally 
it  is  present  as  a  hippurate  to  the  amount  of  0  •  7  grm.  in 
24  hours.  The  quantity  is  markedly  augmented  when  ben- 
zene derivatives  such  as  exist  in  plants  and  fruits  are  inges!  ed. 
An  increase  in  amount  may  also  result  from  excessive  putre- 
faction of  vegetable  material  in  the  intestine.  Thus  it  is  that 
larger  quantities  of  hippuric  acid  are  always  found  in  the 
urine  of  herbivora  than  in  that  of  carnivora.  It  readily  crys- 
tallizes in  long  rhombic  prisms  or  needles  formed  in  rosettes, 
and  is  soluble  in  water  and  alcohol.  If  urine  containing 
hippuric  acid  is  acidified. (20-30  c.c.  of  cone.  HC1  to  the  liter) 
and  evaporated  on  the  water-bath  to  a  small  volume,  crystals 
of  this  substance  will  form  upon  cooling.  They  may  be  col- 
lected on  a  filter  and  washed-  with  a  little  alcohol  saturated 
with  ether. 

(a)  Heat  a  few  crystals  in  a  dry  test-tube.  They  will 
melt  at  187°  C.,  and  at  still  higher  temperatures  will  decom- 


URINE.  99 

pose  with  the  formation  of  a  red  coloration  (decomposition 
of  the  glycocoll)  and  an  odor  resembling  that  of  the  oil  of 
bitter  almonds  (benzonitrile). 

PIGMENTS. 

Variation  in  the  yellow  color  of  the  urine  noticed  under 
certain  conditions,  is  attributable  to  the  presence  of  different 
pigments,  the  most  important  of  which  are  urochrome,, 
urobilin,  and  uroerythrin. 

UROCHROME. 

This  is  the  name  of  the  substance  which  gives  to  normal 
urine  its  characteristic  yellow  color.  Of  its  properties  and 
constitution  next  to  nothing  is  known.  It  is  isolated  from 
urine  when  the  latter  is  saturated  with  (NH4)2S04  and  the 
filtrate  extracted  with  alcohol.  The  pigment  is  soluble 
in  the  alcohol  and  the  entire  color  of  the  urine  passes  into 
the  solvent.  Urochrome,  when  acted  upon  by  mild  reducing 
agents,  yields  a  second  pigment  which  is  apparently  identical 
with  urobilin.  The  name  uroerythrin  is  applied  to  the  pig- 
ment which  imparts  to  the  brick-dust  sediment  of  urates 
(sedimentum  lateritium)  its  pinkish  coloration.  Solutions 
of  this  pigment  are  rapidly  decolorized  by  light. 

UROBILIN. 

Although  not  ordinarily  present  to  such  an  extent  as 
urochrome,  urobilin  is  frequently  increased  in  diseased 
conditions,  and  its  importance  lies  in  the  knowledge  which 
has  been  gained  concerning  its  origin  from  and  relation  to 
the  biliary  pigments.  The  substance  is  extremely  soluble, 
dissolving  in  all  ordinary  solvents.  It  exhibits  in  alcoholic 


100  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

solutions  a  green  fluorescence  which  is  greatly  augmented 
if  ammonia  and  alcoholic  ZnCl?,  solution  are  added.  Acid 
alcoholic  solutions  of  urobilin  also  show  a  distinct  absorption- 
band  between  6  and  F. 

In  constitution  urobilin  possesses  the  same  acidic  proper- 
ties which  characterize  all  the  animal  pigments.  It  forms 
insoluble  compounds  with  bases,  and  is  completely  precipi- 
tated from  its  solution  by  saturation  with  (NH4)2S04  (differ- 
ing from  urochrome). 

(a)  To  50  c.c.  of  urine  faintly  acidified  with  H2S04  add 
ammonium  sulphate  (in  substance)  to  saturation.  Filter  off 
the  precipitate  and  dissolve  it  in  alcohol  containing  a  few 
drops  of  concentrated  HC1.  What  sort  of  a  body  is  urobilin? 

(6)  To  50  c.c.  of  urine  add  an  equal  mixture  of  neutral 
and  basic  lead  acetate  until  the  precipitation  is  complete. 
Filter  and  allow  the  precipitate  to  drain  as  dry  as  possible. 
Then  place  it  and  the  filter-paper  in  an  evaporating-dish 
half  full  of  95  per  cent  alcohol  acidulated  with  HC1.  Warm 
over  the  water-bath  until  the  alcohol  is  well  colored.  Filter 
and  examine  the  solution  in  the  spectroscope.  Now  add  a 
few  drops  of  NH4OH  and  a  few  c.c.  of  an  alcoholic  zinc 
chloride  solution.  Note  the  fluorescence.  In  what  way  is 
the  coloring  matter  of  the  urine  precipitated? 

(c)  Shake  25  c.c.  of  urine  with  2  to  3  drops  of  pure  HC1 
and  5  c.c.  of  chloroform.  Remove  the  chloroform  and 
stratify  upon  it  a  few  c.c.  of  a  solution  of  zinc  acetate  in 
alcohol.  Notice  the  green  ring;  by  shaking,  the  solution 
becomes  fluorescent. 


CARBOHYDRATES  AND  RELATED  BODIES. 

Under    apparently    normal    conditions,    reducing    sub- 
stances of  a  carbohydrate  nature  appear  in  the  urine  in  small 


URINE.  101 

and  variable  quantities.  The  most  important  of  these  are 
the  pentoses,  glycuronates,  and  possibly  dextrose.  It  is 
still  debatable  whether  dextrose  is  present  in  the  urine  under 
absolutely  normal  conditions,  or,  better,  under  conditions 
which  do  not  permit  of  the  slightest  suspicion  that  the  renal 
function  is  impaired.  If  it  does  exist  in  the  urine  in  such 
cases,  it  is  present  in  amounts  which  escape  detection  by  the 
usual  methods. 


PENTOSES. 

As  the  methods  for  isolation  and  identification  of  the 
pentoses  improve,  the  presence  of  these  substances  in  the 
urine  is  being  more  frequently  noted.  Xylose,  Arabinose, 
and  Rhamnose  (methyl-pentose)  have  been  described.  They 
originate  in  the  organism  after  the  ingestion  of  cherries, 
plums,  grapes,  etc.,  which  contain  the  mother  substances 
of  the  pentoses,  the  pentosanes.  The  pentoses  are  not 
readily  assimilable,  and  when  ingested  appear  in  part  un- 
changed in  the  urine.  It  must  be  remembered  that  the 
tests  which  were  given  for  pentoses  (p.  3)  are  not  abso- 
lutely characteristic  for  these  substances,  since  glycuronic 
acid  and  related  bodies  also  react  positively  with  them. 
The  reactions  may  be  used,  nevertheless,  as  confirmatory  tests 
if  the  polarization  and  fermentative  power  of  the  urine  are 
also  determined.  The  pentoses  found  in  the  urine  are  non- 
fermentable  and  inactive  toward  polarized  light,  while  the 
glycuronates  are  Isevogyrate. 

(a)  Test  the  urine  in  the  polariscope. 

(6)  Try  the  orcinol  and  phloroglucinol  tests  with  10  c.c. 
of  urine. 

(c)  Heat  to  boiling  4-5  c.c.  of  Bial's  reagent  and  then 
add  a  few  drops  of  the  urine. 


102  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

GLYCURONIC  ACID. 

Glycuronic  acid  originates  as  a  product  of  the  inter- 
mediary metabolism  of  the  carbohydrate  nucleus  in  the 
protein  molecule.  Ordinarily  it  is  present  in  the  urine  in 
small  amounts  combined  with  phenol,  indoxyl,  and  skatoxyl 
as  conjugate  glycuronates;  but  under  certain  conditions, 
either  as  the  result  of  an  abnormal  increase  in  the  quantity 
of  these  bodies  just  mentioned,  or  the  ingestion  of  aromatic 
substances,  such  as  the  camphors,  chloral,  and  naphthol, 
etc.,  the  conjugate  glycuronates  appear  in  such  quantities 
as  to  give  to  the  urine  a  left-handed  polarization  and  a 
decided  reducing  power  on  Fehling's  solution.  (Some  glycu- 
ronates do  not  reduce.)  Glycuronic  acid  is  dextrogyrate,, 
does  not  ferment,  and  gives  positive  results  with  the  orcinol 
and  phloroglucinol  tests  (see  the  pentoses).  As  glycuronic 
acid  itself  is  never  present  in  the  urine,  and  since  the  gly- 
curonates are  said  to  respond  negatively  to  the  orcinol  test, 
this  test  has  been  used  as  a  means  of  differentiating  between 
the  pentoses  and  the  glycuronates.  This  is  only  true,  how- 
ever, of  the  glycuronates  which  do  not  split  off  the  glycuronic 
acid  component  during  the  performance  of  the  orcinol  test 
(boiling  on  the  water-bath  with  HC1).  Glycuronic  acid 
forms  with  parabromphenylhydrazin  an  osazone  which 
melts  at  from  200-216°  C.  and  which,  if  dissolved  in  pyridin 
and  alcohol,  shows  a  left-handed  rotation  of  7°  25'  or  369° 
(a)D. 

A  Isevogyrate  urine  which,  upon  treatment  with  Fehling's 
solution,  gives  a  yellowish  precipitate  and  partial  reduction 
of  the  copper  may  be  more  than  suspected  of  containing 
conjugate  glycuronates. 

Examine  a  sample  of  urine  in  the  same  way  as  under 
pentoses. 


URINE.  103 

QUANTITATIVE  DETERMINATIONS. 

PHYSICAL  PROPERTIES. 

Determinations  of  certain  physical  properties  of  the 
urine  have  come  into  prominence  of  late  as  a  means  for 
furnishing  additional  data  concerning  the  general  state 
of  equilibrium  which  obtains  in  the  fluid.  The  urine,  being 
in  fact  merely  a  somewhat  dilute  solution  of  organic  and 
inorganic  salts,  must  possess  the  same  properties  and  respond 
to  the  same  physical  laws  which  govern  such  solutions.  The 
most  important  property  whose  determination  is  attempted 
is  that  of  the  depression  of  the  freezing-point  below  that  of 
the  solvent,  water. 

The  law  states  that  the  presence  bf  substances  dissolved 
in  a  solvent  causes  the  freezing-point  of  the  solution  to  be 
lower  than  that  of  the  solvent,  and  that  the  amount  of  the 
depression  is  in  direct  proportion  to  the  number  of  molecules 
•or  (when  dissociation  has  taken  place)  ions  in  the  solution. 

The  depression  of  the  freezing-point,  which  is  represented 
by  the  Greek  letter  J,  is  therefore  dependent  upon  the 
molecular  concentration. 

The  only  substances  which  are  present  in  the  urine  in  a 
quantity  sufficient  to  exert  any  influence  on  J  are  the  urea 
and  sodium  chloride  and  possibly  the  phosphates  and  sul- 
phates, although  the  effect  of  the  latter  two,  even  if  it  did  lie 
outside  of  the  limit  of  experimental  error,  could  not  suffice 
to  cause  differences  which  would  influence  deductions.  The 
other  factors  which  affect  the  J  are  those  which  cause  varia- 
tions in  the  specific  gravity  of  the  urine,  and  it  is  stated  that 
..a  relationship  exists  between  the  two  which  only  varies 
-within  very  small  limits.  A  priori,  this  seems  impossible 


104  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

when  it  is  remembered  that  solutions  of  urea  and  sodium 
chloride  with  the  same  specific  gravity  would  have  widely 
differing  J,  since  urea  is  non-dissociable,  while  sodium  chlo- 
ride in  the  strength  in  which  it  exists  in  the  urine  disso- 
ciates to  a  considerable  degree. 

The  freezing-point  determination  is  made  by  the  use  of 
the  Beckmann  apparatus  (see  demonstration).  The  manip- 
ulation is  simple,  rapid,  and  requires  little  experience. 

Another  property  of  the  urine  which  is  dependent  upon 
the  presence  of  electrolytes  in  the  solution  is  termed  the 
electrical  condiictivity.  Electrolytes  are  substances  which 
become  capable  of  conveying  electricity  in  virtue  of  the  fact 
that  they  become  dissociated  in  solution  into  (electrically) 
active,  positive  or  negative  parts  or  ions.  Therefore  the 
electrical  conductivity  is  in  direct  proportion  to  the  degree 
of  dissociation  or  ionization  of  the  urine.  This  property  is 
unaffected  by  variations  in  the  content  of  urea  in  the 
urine.  Such  determinations  are  accomplished  by  the  use 
of  a  rather  complicated  apparatus  based  upon  the  theory 
of  the  Wheatstone  bridge.  (See  demonstration.)  The  im- 
perfect knowledge  which  exists  in  regard  to  the  conditions 
obtaining  in  the  various  fluids  of  the  body  does  not  permit 
of  exact  interpretations  of  the  results  acquired  by  these 
methods. 

It  must  suffice,  therefore,  to  merely  mention  that  various 
factors  are  derived  from  a  combination  of  the  estimation  of 
the  J  and  that  of  urinary  substances,  such  as  NaCl,  total 
nitrogen,  etc.,  and  that  such  factors  are  constantly  increas- 
ing in  value  as  data  serving  to  present  a  clearer  picture  of  the 
conditions  under  which  a  given  sample  of  urine  may  have 
been  excreted. 


URINE.  105 


ACIDITY  OF  THE  URINE. 

It  is  quite  generally  understood  that  the  degree  of  acidity 
of  the  urine  is  due  to  the  excess  of  the  dihydrogen  (acid) 
over  the  monohydrogen  phosphates,  the  relation  to  the 
total  phosphates  usually  being  about  60  per  cent  of  the 
former  to  40  per  cent  of  the  latter.  Based  upon  this  view, 
the  methods  for  the  quantitative  determination  of  the  acidity 
have  consisted  in  the  estimation  of  the  total  P205  and  the 
P205  present  in  the  form  of  the  acid  phosphates,  and  from 
which  the  degree  of  relative  acidity  of  the  urine  was  derived. 
Many  difficulties  arise  in  the  procedure  for  the  quantitative 
separation  and  determination  of  the  two  types  of  phosphates, 
and  the  present  methods  have  been  shown  to  be  full  of 
errors.  Theoretical  objections  have  also  been  raised,  and 
attempts  made  to  show  that  the ' '  organic  "  acidity,  as  opposed 
to  the  " mineral"  (phosphate)  acidity,  plays  a  not  incon- 
siderable role  in  determining  the  total  acidity.  How  un- 
important the  estimation  of  the  organic  acidity  really  is 
can  be  understood  when  it  is"  noted  that  organic  acids  appear- 
ing in  a  solution  of  mixed  phosphates  are  able  to  remove  the 
base  from  the  monohydrogen  phosphates,  and  thus  pro- 
duce an  almost  equivalent  increase  in  the  quantity  of  the 
acid  phosphates.  The  presence  of  the  organic  acids,  there- 
fore, only  serves  to  increase  the  acid  phosphates,  and  the 
latter  still  remain  theoretically  the  most  accurate  indica- 
tion of  the  total  acidity.  The  technical  difficulties  have 
led  to  the  reliance  upon  the  method  of  direct  titration  of 

the  urine  with  r^  alkali.    This  procedure  has  the  advantage 

of  rapidity  and  possesses  some  value,  perhaps,  where  com- 
parisons only  are  required.  Absolute  values  in  the  present 
state  of  the  question  are  unattainable. 


106  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

Quantitative  Estimation. 

To  25  c.c.  of  urine  in  a  flask  add  15-20  grms.  of  powdered 
potassium  oxalate  and  two  drops  of  phenolphthalein.  Allow 
the  oxalate  to  partly  dissolve  by  shaking  and  titrate  the 

mixture  immediately  with  ^  NaOH  until  the  first  faint 

pink  coloration  is  discernible.     The  acidity  in  this  case  is 

N 
expressed  in  terms  of  j~  acid  for  100  c.c.  of  urine. 


CHLORIDES. 

The  quantity  of  chlorine  excreted  in  the  urine  during  the 
24  hours  is  subject  to  great  variations.  On  a  mixed  diet  it 
usually  amounts  to  from  10  to  15  grms.,  calculated  as  NaCl. 
Under  normal  conditions  the  quantity  eliminated  is  depend- 
ent upon  the  quantity  of  chlorine  and  water  ingested;  but 
in  pathological  states,  such  as  the  formation  and  subsequent 
absorption  of  exudates,  it  is  respectively  markedly  decreased 
and  then  increased.  In  starvation  the  amount  becomes 
minimal. 

Quantitative  Estimation. 

The  principle  of  the  method  is  the  following: 
To  the  urine  is  added  an  excess  of  AgN03  over  and  above 
what  is  necessary  to  precipitate  all  the  chlorides  present. 
The  excess  of  Ag  is  then  determined  by  means  of  a  sulpho- 
cyanide  solution,  using  iron  alum  as  an  indicator. 
Reagents  necessary: 

1.  A  AgN03  solution,  each  c.c.  of  which  precipitates  0.01 
grm.  NaCl  (29.075  grms.  AgNO3  in  a  liter). 

2.  A  saturated  solution  of  iron  alum. 

3.  Chlorine-free  HN03  of  a  specific  gravity  1.2. 


URINE.  107 

4.  A  potassium  sulphocyanide  solution  of  which  2  c.c. 
corresponds  to  1  c.c.  of  the  known  AgN03  solution. 

Method:  Prepare  a  clean  and  dry  graduate.  By  means 
of  a  pipette  measure  off  accurately  5  c.c.  of  urine  and  run  it 
into  the  graduate.  Add  3  c.c.  of  the  HN03  and  dilute  with 
water  to  about  25  c.c.  Then  allow  exactly  10  c.c.  of  the 
known  AgN03  solution  to  flow  in.  Add  water  until  the 
volume  equals  50  c.c. ;  then  mix  thoroughly.  Transfer  this 
now  to  a  clean  and  dry  beaker  and  clean  and  dry  the  gradu- 
ate again.  Prepare  also  a  clean  and  dry  funnel  with  paper 
and  filter  the  mixture  in  the  beaker  into  the  graduate.  When 
25  c.c.  of  a  water-clear  nitrate  has  passed  through  remove  the 
funnel  to  a  test-tube.  Add  10  drops  of  the  iron  alum  to  the 
graduate  and  titrate  with  the  known  potassium  sulphocya- 
nide until  the  first  tinge  of  pink  appears  in  the  solution. 

Calculate  the  amount  of  chlorine  or  Nad  in  the  5  c.c.  of 
the  urine  used,  and  then  in  the  24-hour  sample. 


SULPHATES =S03. 

A  variable  fraction  (80  to  90  per  cent)  of  the  total  sulphur 
of  the  urine  exists  in  a  completely  oxidized  form  as  the  salts  of 
sulphuric  acid,  usually  denoted  and  calculated  as  S03.  Of  the 
total  S03,  about  nine-tenths  is  combined  with  bases  (preformed) 
anc?  one-tenth  with  aromatic  radicles  (conjugate  sulphates). 
The  "organic  "  sulphur  appears  as  taurine,  cystine,  KSCN,  etc. 

Since  the  food  contains  merely  minimum  quantities  of 
S03,  that  found  in  the  urine  must  originate  as  a  product 
of  protein  metabolism,  in  which  the  sulphur  of  the  molecule 
becomes  oxidized.  Thus  it  is  that  ordinarily  the  S03  output 
mav  be  considered  as  indicative  of  the  amount  of  protein 
burned  in  the  body.  About  1.5-3.0  grms.  (average  2.5 
grms.)  SOs  are  excreted  during  24  hours. 


108  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

Quantitative  Estimation. 

The  procedures  for  the  determination  of  these  various 
units  rest  upon  the  following  facts: 

Boiling  the  urine  with  dilute  HC1  liberates  all  the  sul- 
phate radicles  present  in  such  a  form  that  they  may  be 
precipitated  with  BaCl2  as  BaS04.  This  gives  the  amount  of 
total  sulphate.  If  acetic  acid  be  used  instead  of  HC1,  the 
resulting  precipitate  with  BaCl2  will  be  made  up  of  the  pre- 
formed or  sulphate-sulphate.  This  may  be  filtered  off  and 
the  filtrate  treated  as  for  the  total  sulphates,  the  result  being 
the  amount  of  ethereal  sulphates  present.  The  difference 
between  this  and  the  total  will  be  the  amount  of  the  pre- 
formed sulphate. 

TOTAL  S03. 

In  a  beaker  place  50  c.c.  of  filtered  urine  and  dilute  with 
100  c.c.  of  water,  adding  5  c.c.  of  HC1.  Heat  to  boiling,  add 
very  slowly  20  c.c.  of  the  BaCl2  solution  and  allow  it  to  cool 
and  stand  covered  in  a  cool  place  for  24  hours.  Filter  through 
a  small  ash-free  filter;  the  precipitate  must  be  removed  quan- 
titatively from  the  beaker  to  the  paper  by  means  of  warm 
water.  Now  wash  the  white  precipitate  with  water  until  the 
washings  give  no  test  for  chlorine;  then  dry  at  100°  C. 
When  dry,  slip  out  the  paper  from  the  funnel,  fold  it  up  so 
that  the  contained  sulphate  cannot  fall  out  and  place  it  in  a 
porcelain  crucible  which  has  previously  been  ignited  and 
weighed.  Ignite  the  paper  in 'the  crucible  carefully  and 
burn  until  the  residue  becomes  white.  Cool  the  crucible  and 
weigh.  The  difference  in  the  two  weights  will  give  that  of  the 
BaS04  from  50  c.c.  of  urine,  from  which  may  be  calculated 
the  S03  in  the  total  24-hour  sample. 


URINE.  109 


CONJUGATE  S03. 

To  75  c.c.  of  urine  in  a  beaker  add  an  equal  volume  of 
water;  acidulate  with  about  10  drops  of  acetic  acid,  heat 
to  boiling  and  a,dd  10  c.c.  of  the  BaCl2  solution.  Place  the 
covered  beaker  in  a  cool  place  for  about  24  hours.  The 
precipitate  is  then  collected  on  a  filter  and  in  the  filtrate 
and  combined  washings  the  S03  is  determined  as  outlined 
under  total  S03.  In  this  case  the  result  corresponds  to 
the  S03  of  the  conjugate  sulphates. 

ORGANIC  SULPHUR. 

Evaporate  50  c.c.  of  urine  down  to  .dryness,  add  the 
fusion  mixture  and  fuse  with  an  alcoholic  flame  until  a  white 
residue  remains.  Take  this  up  with  hot  water  strongly 
acidulated  with  HC1.  Determine  the  amount  of  S03  present 
in  this  solution  and  calculate  the  amount  for  the  24  hours, 
from  which  may  be  subtracted  the  total  S03  which  has  been 
previously  estimated  in  another  sample  of  the  same  urine. 
The  difference  will  give  the  organic  sulphur  expressed  as 
S03  in  the  24-hour  sample. 

PHOSPHATES  =  P205. 

Phosphorus  is  present  in  the  urine  to  the  greatest  extent 
as  phosphoric  acid  combined  with  bases,  but  a  small  quantity 
(2-2.5  per  cent)  exists  as  organic  P.;  e.g.,  glycerophosphoric 
acid,  lecithin,  etc.  It  has  been  stated  that  the  amount  of 
this  latter  form  of  phosphorus  is  a  true  indication  of  the  ni- 
trogenous metabolism  taking  place  in  the  body.  The  phos- 
phoric acid  phosphorus  is  expressed  as  P205,  and  during 
24  hours  about  1.5-3.5  grms.  (average  2.5  grms.)  P206 


110  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

are  excreted.  This  amount  is  made  up  of  alkaline  and 
earthy  P205  in  decidedly  varying  proportions;  of  the  alkaline 
P205  about  60  per  cent  consists  of  the  dihydrogen  (acid) 
phosphate  and  the  remaining  40  per  cent  as  the  monohydrogen 
phosphate. 

TOTAL  P205. 

The  procedure  is  based  upon  the  quantitative  precipita- 
tion of  all  the  phosphates  in  the  urine  with  uranium  nitrate, 
as  uranium  phosphate,  using  potassium  ferrocyanide  as 
indicator.  The  phosphates  must  be  present  as  acid  salts. 

Quantitative  Estimation. 
Reagents  necessary: 

1.  A  uranium  nitrate  solution  1  c.c.  of  which  equals  0.005 
grm.  P205  (35.461  grms.  in  a  liter).     How  is  this  calculated? 

2.  An  accessory  solution  (100  grms.  of  sodium  acetate  and 
300  grms.  of  acetic  acid  in  a  liter). 

3.  A  solution  of  potassium  ferrocyanide.    What  is  the 
reaction  which  indicates  the  absolute  precipitation  of  all 
the  P205? 

Method:  Place  25  c.c.  of  urine  in  a  large  evaporating-dish, 
add  5  c.c.  of  the  accessory  solution  (why?),  and  warm  gently 
over  an  asbestos  board.  Keeping  the  solution  warm,  add  the 
known  uranium  nitrate  solution  from  a  burette,  from  time  to 
time  removing  a  drop  of  the  urine  on  the  end  of  a  glass  rod 
and  adding  it  to  a  drop  of  the  potassium  ferrocyanide  which 
has  been  placed  upon  a  white  porcelain  dish.  When  all  the 
P205  has  been  precipitated  by  the  uranium  and  the  first  ex- 
cess of  the  latter  appears  in  the  urine  solution,  the  drop  of 
indicator  on  the  plate  when  tested  as  above  will  take  on  a 
faint  reddish-brown  color.  The  test  should  be  repeated  after 
a  minute;  if  it  again  causes  a  color,  the  titration  is  complete, 


URINE.  Ill 

otherwise  more  of  the  uranium  solution  must  be  added  and 
retested. 

From  the  number  of  c.c.  of  the  uranium  nitrate  solution 
required  calculate  the  P205  in  the  25  c.c.  of  urine  employed 
and  from  that  determine  the  total  P205  in  the  24-hour 
urine. 

EARTHY  P205. 

In  the  precipitation  of  the  earthy  phosphates  by  means 
of  NH4OH  various  errors  creep  in  which  render  the  determina- 
tion of  doubtful  value.  Thus,  in  whatever  form  the  earthy 
bases  (Ca  or  Mg)  were  present  in  the  urine,  they  would  form 
insoluble  phosphates  when  the  fluid  was  made  alkaline,  and 
the  precipitate  would  therefore  contain  P205  not  originally 
combined  with  the  alkaline  earths. 

The  method  is  given  because  it  is  still  employed. 

Quantitative  Estimation. 

Place  50  c.c.  of  urine  in  a  beaker  and  make  it  alkaline  with 
NH4OH.  A  precipitation  of  the  earthy  phosphates  occurs. 
Allow  this  to  stand  for  a  couple  of  hours  and  then  collect  the 
precipitate  upon  a  small  filter.  After  having  washed  the 
precipitate  with  very  dilute  NH4OH  transfer  it  quantitatively 
to  an  evaporating-dish  by  means  of  dilute  acetic  acid,  which 
dissolves  the  P205.  Dilute  the  solution  to  about  25  c.c.  and 
titrate  as  outlined  under  total  phosphates.  The  difference 
between  this  amount  and  that  of  the  total  will  be  the  amount 
of  alkaline  P205. 

ORGANIC  PHOSPHORUS. 

The  method  employed  for  the  determination  of  the  organic 
phosphorus  in  the  urine  is  based  upon  the  same  principle  as 
that  in  use  for  organic  sulphur.  The  total  phosphorus  is 


112  LABORATORY  WORK  IX  PHYSIOLOGICAL  CHEMISTRY. 

estimated  after  fusion  as  P205.  From  this  amount  is  de- 
ducted the  total  P205  determined  on  another  sample  of  the 
same  urine,  and  the  difference  corresponds  to  the  organic 
phosphorus  calculated  in  terms  of  P205. 

TOTAL  NITROGEN  (KJELDAHL). 

A  well-nourished  man  eliminates  under  ordinary  con- 
ditions with  a  mixed  diet  10-16  grms.  nitrogen.  The  amount 
is  dependent  upon  the  body  weight,  the  diet,  and  various 
factors  which  may  influence  the  metabolism  of  protein  in  the 
organism.  Muscular  work  has  no  effect  upon  the  nitrogenous 
excretion.  In  round  numbers  85  per  cent  of  the  nitrogen  is 
in  the  form  of  urea;  4  to  5  per  cent,  NH3;  1  to  2  per  cent, 
uric  acid;  and  the  remaining  extractives,  etc.,  8  to  10  per 
cent. 

Quantitative  Estimation. 

The  Kjeldahl  method  for  the  determination  of  the  total 
nitrogen  of  the  urine  has  come  into  almost  universal  use. 

The  principle  consists  in  the  decomposition  of  all  the  or- 
ganic matter  by  heating  with  sulphuric  acid,  whereby  all  of 
the  carbon  and  hydrogen  become  oxidized  to  C02  and  H20, 
and  the  nitrogen  of  such  compounds  which  contain  it  in 
combination  with  hydrogen  (such  as  =NH,  NH2,  NH3), 
but  not  with  oxygen,  appears  as  ammonia.  This  is  lib- 
erated from  the  acid  solution  by  saturation  with  NaOH; 
the  gas  is  then  distilled  over  into  a  known  quantity  of  acid, 
the  amount  of  which  thus  neutralized  being  determined  by 
the  titration  of  the  acid  still  remaining. 

Reagents  necessary   (all  nitrogen-free): 

1.  Concentrated  H2S04   (2  parts  fuming:  3  parts  pure 
concentrated). 

2.  Potassium  sulphate  (powdered). 


URINE.  113 

3.  Solution  of  sodium  hydrate,  sp.  gr.  1.23  (two-thirds 
saturated). 

4.  Solution  of  ^  NaOH. 

N 

5.  Solution  of  ^  H2S04. 

Detailed  Method. — 5  or  10  c.c.  of  urine  (according  to  con- 
centration) are  measured  out  by  a  pipette  and  placed  in  a 
long-neck  digestion  (Kjeldahl)  flask.  To  this  is  added  10  c.c. 
of  the  cone.  H2S04  and  one-half  of  a  teaspoonful  of  potassium 
sulphate.  The  mixture  is  allowed  to  boil  over  a  sand-bath  or 
wire  gauze  until  the  solution  becomes  water-clear  (3  to  6  hrs.). 
After  the  flask  has  cooled,  the  contents  are  removed  quan- 
titatively to  an  Erlenmayer  flask  (content,  1  liter),  using 
about  400  c.c.  of  water,  and  to  this  solution  is  added  without 
mixing  40-50  c.c.  of  the  strong  NaOH  solution.  The  flask 
is  now  quickly  connected  by  means  of  a  rubber  cork  with  a 
condenser-tube,  the  other  end  of  which  is  immersed  in  a  known 

N 
quantity  of  ^  H2S04  (50-200  c.c.  according  to  the  estimated 

amount  of  nitrogen  in  the  urine  employed).    The  vessel 

N 
used  to  hold  the  ^  H2S04  is  usually  a  small  Erlenmayer 

flask.  The  contents  of  the  large  flask  are  well  mixed  and 
a  flame  placed  beneath  a  wire  gauze  upon  which  the  flask 
must  rest.  After  the  beginning  of  ebullition,  the  boiling 
should  be  continued  for  45  minutes.  This  must  be  regulated 
so  that  the  NH3  comes  over  gradually.  At  the  end  of  the 
time  the  small  flask  is  removed  so  that  the  end  of  the  con- 
denser still  remains  in  the  flask  but  is  not  in  contact  with 
the  fluid.  It  must  be  held  in  this  position  for  some  minutes 
to  allow  the  condenser-tube  to  be  washed  inside  with  the 
water  still  distilling  over,  and  outside  with  a  stream  from 


114  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

the  wash-bottle.      Then  turn  oft  the  flame.    The  amount  of 

N  N 

^-r  acid  still  unneutralized  is  titrated  with  ^  NaOH.     The 

difference  between  this  and  the  amount  originally  placed 

N 

in  the  flask  will  represent  the  amount  of  the  ^  acid  neu- 
tralized by  the  NH3  which  was  distilled  over. 

N 

1  c.c.  -JQ  H2S04=  0.0014  grm.  N.,  from  which  may  be 

calculated  the  quantity  of  N.  in  the  10  c.c.  used  and  then 
in  the  24-hour  sample. 


UREA  (HUFFNER'S  METHOD). 

For  the  determination  of  urea,  the  following  method, 
whose  use  has  become  almost  universal,  seems  sufficiently 
accurate  to  warrant  its  continuance  in  favor.  The  rapidity 
with  which  it  can  be  performed  offsets  the  disadvantage 
of  the  mere  approximation  of  results  and  renders  it  pre- 
eminently suited  for  clinical  purposes. 

Only  about  92  per  cent  of  the  nitrogen  of  the  urea  actually 
present  in  the  urine  is  obtained  as  a  gas,  although  the  theoret- 
ical reaction  is  quantitative, 


CO/ 


NH2 

+  3NaOBr=  N2+C02+2H20+3NaBr 
NH2 


(see  experiment  (e)  under  Urea),  but  the  deficit  is  partially 
diminished  by  the  fact  that  other  substances  (e.g.,  uric  acid) 
also  yield  up  some  of  their  nitrogen  as  a  gas  by  the  decompo- 
sition with  hypobromite. 

_  The  more  accurate  methods  (Morner-Sjoqvist  or  Folin's) 
will  be  demonstrated. 


URINE.  115 


Quantitative  Estimation. 

Reagents  necessary: 

Hypobromite  solution  (see  Appendix). 

In  making  use  of  the  Doremus  or  similar  ureometers,  it  is 
advisable  to  so  dilute  the  urine  that  the  content  of  urea 
will  not  exceed  approximately  0.5  per  cent. 

Rinse  the  ureometer  first  with  water  and  then  fill  it  with 
the  hypobromite  solution,  so  that  when  the  apparatus  is  per- 
pendicular and  no  air  is  at  the  top,  the  amount  of  fluid  in 
the  bulb  covers  the  opening  from  it  to  the  upright  tube. 
Then  draw  up  into  the  pipette  the  exact  amount  of  urine, 
and  placing  it  under  the  surface  of  the  solution  with  the  pipette 
tip  well  into  the  space  below  the  upright  tube,  force  the  urine 
out  of  the  pipette  slowly,  noting  that  the  bubbles  generated 
all  pass  upward  into  the  closed  tube.  Care  must  be  taken 
that  the  last  drop  of  urine  is  expelled  from  the  pipette  with- 
out allowing  any  air  to  escape  from  it  also.  Allow  the  reac- 
tion and  collection  of  gas  to  go  on  for  half  an  hour,  then  make 
a  reading  on  the  scale  on  the  areometer  of  the  apparatus. 
The  readings  become  more  accurate  when  the  whole  appara- 
tus is  immersed  in  a  beaker  of  water  in  such  a  manner  that 
the  levels  of  the  fluids  in  the  two  vessels  correspond.  The 
figures  on  the  scale  of  the  instrument  indicate  grammes  of 
urea  in  1  c.c.  of  the  urine. 

URIC  ACID  (HOPKINS-FOLIN  METHOD). 

The  following  method  seems  to  combine  considerable 
accuracy  with  simplicity  and  rapidity  of  performance.  It 
is  based  upon  the  fact  of  the  precipitation  of  the  soluble 
urates  by  saturation  with  ammonium  salts  (Hopkins); 
and  additional  accuracy  has  been  obtained  by  the  previous 
removal  of  certain  substances,  e.g.,  mucoids,  phosphates 


116  LABORATORY  WORK   IN  PHYSIOLOGICAL  CHEMISTRY. 

(Folin).    The  urates  are  finally  titrated  with  -^  potassium 

permanganate . 

Quantitative  Estimation. 
Reagents  necessary: 

1.  500  grms.  (NH4)2S04,  5  grms.  uranium  acetate,  and 
60  c.c.  of  10  per  cent  acetic  acid  dissolved  in  water  and  the 
mixture  made  up  to  a  liter. 

2.  KTT  K2Mn208  titrated  against  a  ^  solution  of  oxalic 

acid. 

Place  300  c.c.  of  urine  in  a  beaker,  add  75  c.c.  of  the 
ammonium  sulphate  reagent  and  mix  thoroughly.  After 
the  resulting  precipitate  has  settled  sufficiently  (5  minutes), 
the  mixture  is  filtered  through  a  double-folded  filter.  When 
250  c.c.  of  the  filtrate  have  passed  through,  this  volume  is 
divided  into  two  portions  of  125  c.c.  each,  to  serve  as  dupli- 
cates. To  each  portion  add  5  c.c.  of  concentrated  NH4OH, 
mix  thoroughly,  and  allow  them  to  stand  for  24  hours.  The 
precipitated  ammonium  urate  is  then  transferred  quanti- 
tatively to  a  filter,  using  a  10  per  cent  (NH4)2SO4  solution 
to  remove  the  last  portions  of  the  precipitate  from  the  beaker. 
After  removing  the  filter-paper  from  the  funnel  and  opening 
it  up,  the  precipitate  is  washed  with  about  100  c.c.  of  water 
into  the  same  beaker  in  which  the  ammonium  urate  was 
precipitated;  to  this  15  c.c.  of  cone.  H2S04  is  added  and  the 

N 
mixture  immediately  titrated  with  the  ^:  K2Mn208  solution, 

which  is  added  from  a  burette  until  the  first  permanent 

N 
tinge  of  pink  color  appears.     1  c.c.  of  ^  K2Mn208  solution = 

3.75  mg.  uric  acid.     Calculated  the  quantity  of  uric  acid  in 
the  24-hour  sample. 


URINE.  117 


AMMONIA. 

Ammonia  in  the  form  of  ammonium  salts  is  present  in 
the  urine  of  carnivora  to  the  extent  of  0.3-1.2  (average  0.7) 
grms.  for  24  hours.  Its  function  in  the  body  consists  in 
combining  with  and  thus  rendering  non-toxic  the  mineral 
and  organic  acids  which  appear  in  the  organism,  either  after 
their  ingestion  or  their  formation  in  metabolism.  The 
amount  found  in  the  urine  is,  therefore,  markedly  increased 
in  certain  pathological  states  (e.g.  diabetes)  in  which  more 
or  less  large  quantities  of  organic  acids  make  their  appear- 
ance in  the  blood  as  a  result  of  disordered  metabolism. 

Quantitative  Estimation. 

Prepare  two  500-cc.  wide-mouthed  Erlenmeyer  flasks  and 
one  tall  cylinder  (100  c.c.  graduate) ;  into  the  neck  of  each 
insert  a  two-hole  rubber  stopper,  one  hole  of  which  is  pro- 
vided with  an  L-shaped  glass  tube,  each  arm  being  about  3 
inches  long,  the  other  hole  provided  with  an  L  tube,  the  long 
arm  extending  to  within  one-quarter  inch  of  the  bottom  of 
the  vessels.  A  Folin  absorption  tube  may  be  employed  in- 
stead of  the  long  arm  in  the  absorption  flasks. 

Place  25  c.c.  of  urine  in  the  tall  cylinder  with  about  one 
gram  of  dry  sodium  carbonate;  cover  the  urine  with  one-half 
inch  of  crude  petroleum  to  prevent  foaming. 

To  each  flask  add  20  c.c.  ^  H2S04,  200  c.c.  of  distilled 

water  and  a  few  drops  of  an  indicator  (Congo  red  or  alizarin 
red).  Place  the  three  bottles  in  series  with  the  cylinder  in 
the  middle,  and  connected  in  such  a  manner  that  the  short 
arm  of  one  bottle  is  attached  to  the  long  arm  of  the  succeeding 
one.  The  long  arm  of  the  first  flask  is  open  to  the  air;  the 
short  arm  of  the  last  bottle  must  be  connected  with  a  suction 
pump.  The  purpose  of  the  first  flask  is  to  insure  the  passage 


118  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

of  ammonia-free  air  into  the  cylinder;  the  acid  in  the  second 
flask  absorbs  the  ammonia  liberated  by  the  urine  and  carried 
over  by  the  current  of  air  sucked  through  the  system.  Suc- 
tion should  be  continued  for  about  two  hours. 

N 
The  collection  flask  should  finally  be  titrated  with  ^  NaOH 

and  the  amount  necessary  for  neutralization  subtracted  from 
the  number  of  cubic  centimeters  of  acid  originally  taken. 
The  remainder,  when  multiplied  by  0.0017,  gives  the  amount 
(grams)  of  ammonia  in  25  c.c.  of  urine. 

PROTEIN. 

Quantitative  Estimation. 

Of  the  numberless  methods  which  have  been  suggested 
for  the  quantitative  determination  of  the  total  protein  in 
the  urine,  the  following  has  received  perhaps  the  most  marked 
approbation  from  the  clinicians.  It  is  based  upon  the  quan- 
titative precipitation  by  picric  acid  in  the  presence  of  an 
organic  acid,  of  all  forms  of  proteins  which  appear  in  the 
urine  under  abnormal  conditions.  The  results  obtained 
are  sufficiently  accurate  if  the  urine  does  not  contain  over 
0.4  per  cent  protein.  When  more  than  this  is  present,  the 
urine  must  be  diluted  with  water  in  such  amounts  as  to  allow 
the  percentage  to  fall  below  that  figure. 

The  procedure  is  as  follows : 

Fill  the  albuminometer  to  the  mark  "u"  with  urine  acidi- 
fied with  a  few  drops  of  dilute  acetic  acid,  and  add  Esbach's 
reagent  to  "R  ".  Stop  the  end  of  the  tube  with  a  cork.  By 
inverting  several  times  the  contents  of  the  tube  can  be  thor- 
oughly mixed  without  producing  any  froth  on  the  top  of  the 
mixture.  Allow  the  corked  tube  to  stand  upright  for  24 
hours  and  then  read  off  the  height  of  the  precipitate  on  the 
scale,  which  indicates  directly  the  number  of  grammes  of  dry 
protein  contained  in  a  liter  of  the  urine. 


URINE.  U9 

DEXTROSE. 

All  of  the  methods  for  the  quantitative  estimation  of 
dextrose  in  the  urine  are  founded  upon  the  following  proper- 
ties of  this  carbohydrate: 

1.  Reducing  action  on  copper  and  bismuth. 

2.  Effect  on  the  plane  of  polarized  light. 

3.  Fermentation  by  yeast. 

1.  The  methods  of  this  type  assume  that  the  total  reducing 
power  of  the  urine  may  be  ascribed  to  dextrose.  This  is 
probably  true  in  most  instances,  but  the  fact  of  the  pos- 
sible presence  in  appreciable  quantities  of  other  copper- 
reducing  bodies  must  not  be  neglected  in  critical  examina- 
tions, especially  when  the  reduction  is  small  and  at  the  same 
time  atypical. 

The  Allihn  method  is  by  far  the  most  accurate  in  results 
and  satisfactory  in  its  performance.  Its  accuracy,  how- 
ever, is  entirely  dependent  upon  the  strict  attention  with 
which  all  the  details  of  the  procedure  are  followed  out.  The 
cuprous  oxide  produced  by  the  reducing  action  of  an  unknown 
amount  of  dextrose  upon  a  definite  volume  of  Fehling's 
solution  is  filtered  off  and  further  reduced  to  metallic  cop- 
per by  a  stream  of  hydrogen  gas.  This  metallic  copper  is 
then  weighed.  The  quantity  of  sugar  which  corresponds 
to  this  weight  of  copper  is  found  upon  consultation  of  tables. 
The  details  are  too  numerous  to  allow  of  their  explanation 
in  a  limited  space,  but  the  method  is  to  be  recommended 
highly. 

The  most  common  clinical  method  depends  upon  the 
complete  reduction  as  indicated  by  the  entire  loss  of  color, 
of  a  given  quantity  of  Fehling's  solution,  by  means  of  titra- 
tion  with  diluted  urine. 


120  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

Method:  10  c.c.  of  quantitative  Fehling's.  solution  are 
placed  in  an  evaporating-dish  and  diluted  with  40  c.c.  of 
water.  The  solution  is  brought  to  boiling  and  kept  so  while 
the  urine  (usually  diluted  ten  times)  is  run  in  slowly  from 
a  burette,  until  the  blue  color  of  the  copper  solution  has. 
entirely  disappeared.  An  accurate  determination  of  the  end 
point  of  the  reaction  requires  considerable  experience. 

Since  10  c.c.  of  Fehling's  solution  are  completely  reduced 
by  0.05  grm.  of  dextrose,  the  quantity  of  urine  required 
to  produce  the  end  reaction  must  contain  that  quantity  of 
dextrose.  From  this  can  be  easily  calculated  the  total 
amount  of  dextrose  in  the  24-hour  urine. 

2.  The  possible  simultaneous  appearance  in  the  urine 
of  laBvogyrate  bodies  (e.g.  glycuronates,  /?-oxybutyric  acid) 
renders  the  application  of  methods  of  this  type  of  doubtful 
value.     Even  when  substances  of  this  kind  are  apparently 
absent,   comparative  values  obtained  by  polarization  and 
reduction  methods  seldom  agree  within  wide  limits.     When- 
ever polarization  determinations  are  made,  they  must  be- 
followed  by  a  second  estimation  after  fermentation. 

The  urine  may  be  decolorized  by  the  addition  of  a  crystal 
or  two  of  lead  acetate  and  subsequent  stirring  and  filtration 
(see  Polarization,  p.  5). 

3.  For  rough  approximations  this  method,  using  Einhorn's 
saccharometer,  serves  the  purpose  better  than  any  other.. 
The  procedure  is  described    under    Monosaccharides,  p.  5. 
A  method  of  this  type  has  also  been  suggested,  based  upon 
the  difference  of  the  specific  gravity  of  urine  observed  before 
and  after  fermentation. 


URINE.  121 


PATHOLOGICAL  URINARY  CONSTITUENTS. 

PROTEINS. 

Representatives  of  nearly  every  class  of  proteins  have 
been  detected  in  urine  at  various  times.  Normal  urine  un- 
doubtedly contains  traces  of  these  substances;  thus,  for 
example,  in  the  "mucous  cloud"  which  separates  out  from 
many  urines  upon  standing  a  phosphoprotein  can  be  demon- 
strated. Of  the  proteins  which  appear  under  well-defined 
morbid  conditions,  the  most  important  are  the  albumins, 
the  globulins,  and  the  proteoses.  So-called  peptone  has 
also  been  noticed.  Since,  however,  the  conception  of  the 
properties  of  the  mixture  which  passes  under  this  name  is 
so  ill-defined  and  changeable,  it  is  probably  better  in  the 
future  to  dismiss  the  term  peptonuria.  Haemoglobin  and 
related  substances  may  also  escape  from  the  blood  into  the 
urine. 

ALBUMIN  AND  GLOBULIN. 

Serum  albumin  and  serum  globulin  usually  appear  to- 
gether in  the  urine  under  the  name  of  albuminuria;  but 
concerning  the  relative  quantities  of  the  two  bodies  under 
the  various  conditions  very  little  is  known.  The  two 
proteins  allow  of  separation  in  the  same  manner  as  was 
employed  under  Blood  Proteins,  p.  73. 

In  testing  urines  suspected  of  containing  protein,  the 
fluid  should  always  be  perfectly  clear.  This  can  be  accom- 
plished either  by  repeated  filtration  through  paper  or  asbestos 
or  by  shaking  with  magnesia. 

The  following  tests  are  best  suited  for  ordinary  conditions: 

(a)  Heat  Test. — Heat  5  c.c.  of  clear  urine  to  boiling  and  add 


122  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

1  to  3  drops  of  dilute  acetic  acid.  If  the  urine  contains  more 
than  a  small  amount  of  albumin,  this  will  settle  out  in  flocks 
after  the  addition  of  acid.  When  mere  traces  are  present  the 
solution  may  only  become  turbid  and  should  then  be  com- 
pared to  the  urine  before  heating,  but  to  which  the  same 
amount  of  acid  has  been  added.  This  test  is  only  of  value  as 
a  positive  one.  When  a  negative  result  is  obtained  other 
tests  should  be  tried  with  a  view  to  confirmation. 

In  some  cases  the  addition  of  acetic  acid  to  boiled  urine 
may  give  rise  to  a  faint  precipitate  or  turbidity  which  disap- 
pears upon  shaking.  This  is  caused  by  the  formation  of  acid 
albumin,  which  may  be  salted  out  by  one-third  saturation 
with  NaCl  after  the  addition  of  more  acetic  acid  to  keep 
the  phosphates  in  solution.  Faintly  alkaline  or  amphoteric 
urine  may  sometimes  give  on  heating  a  precipitate  due  to 
phosphates,  which  is  sometimes  difficult  to  distinguish  from 
a  precipitate  of  albumin.  Again,  such  a  urine  may  remain 
perfectly  clear  and  still  contain  albumin.  The  addition  of  a 
small  amount  of  acid  will,  in  the  first  case,  dissolve  the 
phosphates,  and,  in  the  second,  precipitate  any  albumin 
remaining  soluble.  Phosphoproteins  nor  proteoses  do  not 
react  similarly,  since  both  bodies  are  soluble  in  hot  acid 
solutions;  but  a  precipitate  settling  out  upon  cooling  may 
point  to  the  presence  of  such  substances.  Resins  resulting 
from  the  administration  of  petroleum,  turpentine,  oil  of 
sandal-wood,  tolu-balsam,  etc.,  may  be  present  in  the  urine, 
and  if  so,  will  be  precipitated  by  the  acid.  Such  precipitates 
easily  dissolve  in  alcohol. 

(6)  Heller's  Test.— Place  5  c.c.  of  cone.  HN03  in  a  test-tube 
and  allow  a  few  c.c.  of  the  urine  which  is  being  filtered  to  flow 
from  the  funnel  down  the  sides  of  the  tube.  The  urine  will 
thus  stratify  itself  on  top  of  the  acid,  and  at  the  surface 
of  contact  of  the  two  liquids  the  albumin  will  be  precipitated 


URINE.  123 

and  will  appear  as  a  white  ring.  As  the  HN03  upon  standing 
diffuses  upwards  into  the  urine  the  ring  may  become  broader. 
This  test  is  delicate  enough  for  ordinary  clinical  purposes, 
but  will  not  show  the  presence  of  traces.  Urines  containing 
an  excessive  amount  of  urea  may  form  a  crystalline  precip- 
itate in  this  test,  but  such  a  precipitate  cannot  be  confused 
with  albumin.  Colored  rings  may  also  form,  due  to  the 
oxidation  of  the  urinary  pigments,  and  a  blue  coloration  is 
produced  by  indican.  Substances  mentioned  under  the  heat 
test,  such  as  resins,  etc.,  may  form  a  cloud,  but  simple  tests 
such  as  those  indicated  will  exclude  them. 

(c)  Roberts'  Modification  of  Heller's   Test— This  test  is 
performed  similarly  to  the  last  by  the  stratification  of  a  few 
c.c.  of  the  urine  upon  5  c.c.  of  Roberts'  reagent.     It  has 
some  optical  advantages  and  colored  rings  never  appear  in  its 
use. 

(d)  Acetic  Acid  and  Potassium  Ferrocyanide  Test. — Acidify 
5  c.c.  of  urine  with  two  drops  of  acetic  acid  and  add,  drop  by 
drop,   a  dilute  solution  of  K4FeCN6.     In  the  presence  of 
albumin  a  white  precipitate  occurs  which  dissolves  in  a 
large  excess  of  the  reagent.     Traces  of  albumin  may  be  de- 
tected with  this  reaction.    Should  the  precipitate  dissolve 
upon  heating,  proteoses  may  be  suspected.     The  presence  of  a 
considerable  amount  of  mucin  or  phosphoprotein  in  the  urine 
may  sometimes  give  rise  to  a  precipitate  with  acetic  acid 
alone.     This  must  be  removed  by  filtration  before  the  ferro- 
cyanide  test  can  be  completed. 

(e)  Trichlor acetic  Acid  Test. — Stratify  a  few  c.c.  of  a  con- 
centrated aqueous  solution  of  this  reagent  with  5  c.c.  of 
urine.     A  white  ring  sharply  defined  indicates  the  presence 
of  albumin.     The  precipitate  may  also  be  proteoses,  but  in 
this  case  the  ring  dissolves  with  cautious  warming.     This  test 
is  more  delicate  than  Heller's,  and  by  its  use  smaller  quanti- 


124  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

ties  of  albumin  are  demonstrable  in  urines  with  which  the 
more  common  tests  yield  negative  results. 

(/)  Spiegler's  Test. — Stratify  5  c.c.  of  urine  which  has 
been  slightly  acidified  with  acetic  acid,  upon  a  few  c.c.  of 
Spiegler's  reagent.  In  the  presence  of  albumin  a  white  ring 
appears  at  the  line  of  contact  of  the  two  liquids.  This 
test  is  very  sensitive,  showing  albumin  in  a  dilution  1 : 250,000. 
In  fact  most  normal  urines  indicate  protein  with  the  reagent. 
This  must  be  borne  in  mind  in  making  deductions  from 
its  use.  Urines  containing  iodides  give  a  precipitate  of  HgL. 

(g)  Tanret's  (Bouchardai)  Test. — This  reagent  is  added, 
drop  by  drop,  to  5-10  c.c.  of  the  urine  until  a  turbidity  or 
precipitate  appears.  The  reagent  precipitates  besides  al- 
bumin, mucin,  peptone,  and  alkaloids.  In  cases  where  the 
presence  of  alkaloids  in  the  urine  is  suspected,  the  peptone 
and  alkaloids  may  be  dissolved  in  potassio-mercuric  iodide 
and  the  solution  shaken  out  with  ether,  whereby  the  alkaloid 
is  dissolved. 

PROTEOSES. 

Non-coagulable  bodies  which  are  precipitated  by  satura- 
tion with  (NH4)2S04  and  which  give  the  biuret  reaction, 
have  been  demonstrated  and  isolated  from  urines  under 
different  conditions.  These  substances  react  positively  to 
nearly  all  of  the  proteose  reactions,  and  it  seems  definitely 
decided  that  true  proteoses  actually  appear  at  certain  times 
in  the  urine.  Of  especial  interest  is  the  presence  of  an 
albumose-like  substance  (Bence-Jones'  body)  associated  with 
multiple  myelomata  of  the  bone. 

The  identification  of  these  bodies  follows  from  the  use  of 
the  same  tests  employed  under  Digestion,  p.  54. 


URINE.  125 


DEXTROSE. 

Before  testing  for  dextrose  in  the  urine,  protein,  if  present, 
must  be  removed  by  heat  and  acetic  acid.  The  following 
tests  depend  upon  the  power  of  dextrose  to  reduce  metallic 
oxides,  as  evidenced  by  the  formation  of  precipitates  or  color 
changes.  It  must  be  remembered  that  the  urine  may  also 
contain  other  bodies  such  as  creatinine,  uric  acid,  allantoin, 
hydroquinol,  alkaptonic  acid,  urine  and  bile  pigments,  and 
conjugate  glycuronic  acids,  which  also  reduce  metallic 
oxides  to  a  slighter  degree,  however.  It  is,  therefore,  better 
never  to  base  a  decision  entirely  upon  reduction  tests. 

Never  allow  the  urine  to  boil  more  than  a  few  seconds  in 
performing  the  tests.  This  will  tend  to  eliminate  the  possi- 
bility of  a  reduction  caused  by  the  above-mentioned  sub- 
stances. 

(a)  Trommer's,  Fehling's,  and  Fermentation  Tests. — Per- 
form these  tests  as  suggested  under  Monosaccharides  (p.  4), 
using  the  urine  instead  of  the  dextrose  solution. 

(6)  Benedict's  Modification  of  Fehling's  Test. — To  5  c.c.  of 
Benedict's  solution  add  five  to  eight  drops  of  urine.  Boil 
the  mixture  vigorously  for  one  or  two  minutes  and  allow  the 
test  tube  and  contents  to  cool  on  the  rack.  A  precipitate 
will  form  in  the  solution,  the  color  of  which  may  be  red, 
yellow,  or  green,  according  to  the  amount  of  dextrose  present. 
This  test  is  more  sensitive  than  Fehling's  and  has  the  advantage 
that  the  test  solution  does  not  deteriorate  upon  longstanding^ 

(e)  Nylander's  Test. — To  10  volumes  of  urine  add  1  volume 
of  Nylander's  reagent  and  heat.  The  presence  of  sugar  is 
indicated  by  a  dark  coloration  of  the  urine  followed  by  a 
separation  of  a  black  precipitate.  By  this  very  sensitive  test 
the  reducing  character  of  some  normal  urines  may  be  shown. 


126  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

Urines  containing  sulphides  cannot  be  tested  by  this  method. 
Why? 

(d)  Phenylhydrazin  Test. — Perform  this  test  as  given  under 
the  Monosaccharides,  using  15  c.c.  of  urine.     As  other  sub- 
stances in  the  urine  may  give  a  precipitate  with  the  reagent, 
a  mere  separation  of  an  insoluble  body  is  not  sufficient 
evidence  for  the  presence  of  a  sugar.    The  precipitate  must 
be  yellow  and  must  be  examined  carefully  and  critically 
under  the  microscope.    If  sufficient  quantities  are  obtainable 
for  a  melting-point  determination,  this  procedure  should  be 
carried  out.     Phenylglucosazone  melts  at  204-205°  C. 

(e)  Polarization  Test. — As  it  was  stated  in  the  discussion 
of  the  value  of  this  test  for  quantitative  purposes,  the  results 
obtained  are  apt  to  be  misleading,  especially  when  no  polar- 
ization of  the  urine  can  be  detected.    When  taken  as  evidence 
confirmatory  of  other  tests  it  may  serve  a  useful  purpose.    The 
dextro-rotation  must  completely  disappear  aftei  fermentation. 


BILE. 

The  presence  of  bile  imparts  to  the  urine  a  saffron  color, 
•which  upon  standing  takes  on  a  greenish  tinge.  Icteric 
urine  is  usually  cloudy  or  turbid  and  the  sediment,  if  any,  is 
rather  strongly  colored. 

In  order  to  insure  a  positive  identification  of  icteric  urine 
by  means  of  the  bile  acids  it  is  necessary  to  separate  them 
from  the  urine  by  a  long  and  laborious  procedure  and  then  to 
perform  Pettenkofer's  test.  Though  this  is  the  more  accurate 
method,  still  it  is  more  usual  clinically  to  perform  tests  for 
the  presence  of  the  biliary  pigments  directly  on  the  urine. 

(a)  Perform  Gmelin's,  Smith's,  Hammarsten's,  and  Hup- 
pert's  tests. 

(6)  Rosenbach's    Modification    of  Gmelin's    Test.— Filter 


URINE.  127 

some  icteric  urine  and  to  the  moistened  paper  add  one  drop  of 
HN03.  Colored  rings  around  the  drop  correspond  in  color 
and  arrangement  to  those  obtained  in  Gmelin's  test.  Cau- 
tion :  Impure  filter-paper  may  give  this  test,  so  make  a  con- 
trol test  in  using  strange  paper. 

(c)  Haycraft's  Test.— Fill  a  test-tube  half  full  of  fresh 
urine  and  sprinkle  on  the  surface  powdered  sulphur.  If  the 
sulphur  sinks  to  the  bottom  the  presence  of  bile  salts  is  indi- 
cated. This  can  only  be  used  as  a  confirmatory  test. 

BLOOD  AND  BLOOD  PIGMENTS. 

Blood,  as  such,  may  be  present  in  the  urine  (haBmaturia). 
In  these  cases  the  urine  is  cloudy,  brownish-red  in  color,  and 
contains  serum  albumin  and  serum  globulin.  Upon  microscop- 
ical examination  blood  corpuscles  are  found  in  the  sediment. 

In  ha3moglobinuria  the  form  elements  are  absent  and 
the  urine  holds  the  oxyhsemoglobin  in  solution;  metha3- 
moglobin  often  accompanies  it,  and  hsematin  appears  under 
some  conditions.  In  a  number  of  diseases,  but  especially 
after  the  use  of  sulphonal  and  similar  therapeutic  agents, 
hsematoporphyrin  has  been  observed  in  large  quantities. 
Reduced  hemoglobin  is  never  present. 

OXYH^EMOGLOBIN. 

(a)  Notice  the  color  of  the  urine.  If  fresh  it  has  a  reddish 
tinge  and  is  turbid.  Try  the  benzidine  reaction,  (g)  p.  73. 

(6)  Examine  it  with  the  spectroscope.  If  the  urine  is  not 
fresh  be  on  the  lookout'  for  metha3moglobin.  Warm  a  por- 
tion of  the  urine  with  an  excess  of  NaOH;  filter  and  add  a 
few  drops  of  (NH4)2S.  Oxy haemoglobin  is  changed  to  hasmo- 
chromogen  with  its  characteristic  spectrum. 

(c)  Heller's  Test  — Make  the  urine  strongly  alkaline  with 


128  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

NaOH  and  heat  it.  The  oxyhamoglobin  is  split  into  hsematin 
and  protein,  and  the  earthy  phosphates  being  precipitated, 
drag  down  the  hsematin  with  them.  Filter  off  the  precipi- 
tate, which  should  be  of  a  brownish  color,  and  after  drying  it, 
try  to  obtain  Teichmann's  crystals  (p.  76). 

(d)  Struve's  Test. — Make  a  portion  of  the  urine  alkaline 
with  NaOH  and  precipitate  with  tannic  acid.  Test  this  pre- 
cipitate for  hsemin. 


METH^EMOGLOBIN. 

Examine  the  urine  with  the  spectroscope,  add  a  few  drops 
of  NH4OH  and  filter;  a  two-banded  spectrum  appears  similar 
to  that  of  oxyhsemoglobin.  Do  not  confound  the  spectra  of 
methsemoglobin  and  of  hsematin  in  acid  solution. 


HvEMATIN. 

1.  Examine  in  the  spectroscope.     If  a  single  band  is 
present  add  (NH4)2S  to  the  urine,  filter,  and  again  examine  it. 
The  two  bands  of  reduced  hsematin  should  appear. 

2.  Make  tests  c  and  d  under  Oxy hemoglobin. 


ILEMATO  PORPHYRIN. 

The  preparation  of  this  substance  from  the  urine  for  the 
purposes  "of  identification  and  spectroscopic  examination  is 
as  follows: 

Method  of  Garrod.—To  100  c.c.  of  urine  add  20  c.c.  of 
10  per  cent  NH4OH.  The  phosphates  of  the  earthy  metals 
are  precipitated  and  with  them  the  haematoporphyrin.  This 
precipitate  is  filtered  off,  washed,  and  warmed  in  a  flask  with 
acidulated  alcohol.  The  pigment  goes  into  solution.  Use 


URINE  129 

this  for  the  spectroscope.  Upon  the  addition  of  a  small 
amount  of  water  the  alcoholic  extract  will  exhibit  a  red 
fluorescence.  For  tests  see  under  Blood,  p.  78. 


CH2-S—  S-CH2 


CYSTINE,  CH-NH2    CH-NH,. 


This  substance  has  come  into  prominence  of  late  as  a 
protein  decomposition  product  containing  sulphur  in  the 
neutral  or  lead-blackening  form.  In  fact,  the  cystine  which 
can  be  obtained  in  the  decomposition  of  certain  proteins 
contains  an  amount  of  lead-blackening  sulphur  which  corre- 
sponds closely  to  the  total  sulphur  of  the  original  protein 
molecule.  This  would  seem  to  imply  that  the  sulphur  of 
some  proteins  was  present  in  the  molecule  as  a  cystine 
nucleus,  and  that  proteins  containing  large  amounts  of 
neutral  sulphur  have  present  large  numbers  of  cystine  groups 
in  the  molecule.  Cystine  is  also  closely  related  to  Taurine, 


CH2.S03H 
NH2 


•^A-^^ 

t. 


a  substance  which  contains  oxidized  sulphur  in  organic  form 
and  which  probably  represents  an  intermediate  stage  of 
oxidation  between  the  neutral  sulphur  atom  of  cystine  and 
the  mineral  sulphates  of  the  urine.  Cystine,  upon  standing, 
separates  out  of  the  urine  as  colorless  six-sided  plates,  insolu- 
ble in  water,  acetic  acid,  alcohol,  and  ether.  It  sometimes  is 
found  in  the  form  of  calculi.  Cystineuria  has  repeatedly 
been  observed  as  an  apparently  anomalous  metabolism  of 


130  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 

the  sulphur,  peculiar  to  certain  families,  all  the  members  of 
which  excrete  normally  relatively  large  quantities  of  the  sub- 
stance (0.5-1.0  grm.  for  24  hours). 

The  appearance  of  the  characteristic  crystals  and  an 
abnormal  amount  of  lead-blackening  sulphur  in  the  urine 
is  sufficient  proof  of  the  presence  of  cystine. 

FATTY  ACID  DERIVATIVES. 

Associated  with  certain  peculiar  metabolic  disturbances, 
three  closely  related  substances  (/9-oxybutyric  acid,  diacetic 
acid,  and  acetone)  may  appear  in  the  urine  either  separately 
or  together.  They  have  been  noticed  in  severe  cases  of 
diabetes,  scarlet  fever,  cachexia,  etc.,  and  are  the  cause  of 
that  aromatic  fruity  (apple-like)  odor  which  is  so  frequently 
present  in  diabetic  urines.  /3-oxybutyric  acid  only  appears 
in  conjunction  with  acetone  or  diacetic  acid,  but  the  latter 
two  are  often  found  alone  in  the  urine. 


^-OXYBUTYRIC  ACID,  CH3-CH(OH)  -C 

It  is  only  necessary  to  test  for  this  substance  in  urine 
which  contains  diacetic  acid.  Since  this  body  is  Ia3vogyrate 
and  non-fermentable,  the  urine  after  fermentation  should 
turn  the  plane  of  polarized  light  to  the  left.  This  is  not 
sufficient  evidence,  however,  since  the  conjugate  glycuronates 
also  are  Ia3vogyrate  after  fermentation.  The  following  test 
had  better  be  added  as  confirmatory  of  #-oxy  butyric  acid. 

Evaporate  some  fermented  urine  to  a  syrup  and  after 
the  addition  of  an  equal  volume  of  concentrated  H2S04 
distil  directly  without  cooling;  a-crotonic  acid  CH3—  CH 

is    formed    and   distils   over,  and    the    acid 


URINE.  131 

crystallizes  out  from  the  distillate.    The  crystals  are  soluble 
in  ether  and  melt  at  72°  C. 


CH2-COOH 
DIACETIC  ACID,        | 

0=C-CH3 

The  urine  must  be  tested  soon  after  voiding,  as  this  body 
disappears  upon  standing. 

Strongly  acidify  some  urine  with  H2S04  and  shake  it  out 
with  ether.  Separate  the  ether  from  the  solution  and  shake- 
out  the  former  with  water  which  is  just  colored  with  Fe2Cl6. 
The  watery  solution  becomes  violet  and  upon  the  addition  of 
more  Fe2Cle  turns  bordeaux-red. 

The  ferric  chloride  solution  may  also  be  added  to  the  urine 
directly.  In  this  case  the  phosphates  must  be  completely 
removed  by  the  Fe2Cl6  and  filtration.  Then  add  more  Fe2Cl6, 
to  the  filtrate. 

Salicylic  acid  and  salicylates  give  a  similar  reaction. 
When  these  bodies  are,  present  they  may  be  removed  from 
the  acidified  urine  by  shaking  it  out  with  chloroform  or  ben- 
zene in  which  the  diacetic  acid  is  not  soluble.  The  urine  is 
then  treated  with  ether  as  above. 


CH3 
ACETONE,         | 

0=C-  CH3 

Distil  100  c.c.  of  urine  to  which  has  been  added  2  c.c.  of 
50  per  cent  acetic  acid.  Take  the  first  50  c.c.  of  the  distillate,, 
add  1  c.c.  of  concentrated  H2S04  diluted  8  times  and  redistil 
over  25  c.c.  Make  the  following  tests  with  this  solution: 

(a)  Lieben's  Test. — Place  some  of  the  solution  in  a  test- 
tube  and  make  it  alkaline  with  sodium  carbonate.  Add 


132  LABORATORY  WORK  IN  PHYSIOLOGICAL  CdE.tliX 

enough  iodo-potassium  iodide  to  give  the  solution  a  decided 
yellow  color  Warm  at  65°  C.  for  5  minutes  and  allow  to 
cool.  A  yellow  precipitate  of  iodoform  settles  out,  recogniza- 
ble by  its  odor  and  its  hexagonal  crystals.  This  test  is  also 
given  by  alcohol. 

(6)  Gunning's  Modification. — In  this  test  ammonia  is 
substituted  for  the  sodium  carbonate  and  a  tincture  of  iodine 
in  place  of  the  iodopotassium  iodide.  A  black  precipitate  of 
iodide  of  nitrogen  is  first  formed,  but  this  gradually  disappears 
on  standing,  leaving  the  iodoform  visible,  if  present.  Alcohol 
or  aldehydes  do  not  give  this  reaction. 

(c)  LegaVs  Test. — To  a  few  c.c.  of  the  distillate  add  a  few 
drops  of  a  freshly  prepared  solution  of  sodium  nitroprusside 
and  make  the  solution  alkaline  with  NaOH.     A  ruby-red 
color  is  produced  which  quickly  disappears.     Creatinine  also 
gives  this  reaction.     If  the  alkaline  acetone  solution  is  treated 
with  a  large  excess  of  acetic  acid,  the  color  becomes  red, 
whereas  in  the  case  of  creatinine  it  is  changed  to  green  and 
then  blue. 

(d)  Lange's  Modification  of  LegaVs  Test. — To  15  c.c.  of 
urine  add  one  c.c.  of  glacial  acetic  acid,  and  two  or  three  drops 
of  a  freshly  prepared  solution  of  sodium  nitroprusside.     Mix 
this  solution  well  and  carefully  stratify  upon  it  concentrated 
ammonium  hydroxide.     At  the  line  of  contact  a  violet  ring 
will  appear.     This  reaction   is   not   given  with  creatinine, 
alcohols  or  aldehydes. 

EHRLICH'S  DIAZO  REACTION. 

Diazobenzenesulphonic  acid  comes  into  prominence  as  a 
reagent  in  various  connections. 

It  is  employed  in  the  detection  of  sugar,  protein,  bilirubin, 
and  an  unknown  chromogen  of  the  urine  which  is  so  com- 
monly present  associated  with  certain  pathological  conditions 
(typhoid,  pulmonary  tuberculosis,  etc.).  It  is  especially 
as  a  test  for  this  unknown  substance  or  substances  that 


URINE.  133 

diazobenzenesulphonic  acid  was  used  by  Ehrlich  and  has 
since  been  designated  by  the  clinicians  as  Ehrlich's  Diazo- 
reac  ion.  The  fact  must  not  be  forgotten,  however,  that,  as 
a  test  for  bilirubin  this  reagent  has  also  been  employed  by 
Ehrlich,  and  by  others  to  determine  the  presence  of  sugar 
and  protein  in  the  urine.  The  color  obtained  in  the  reaction 
in  the  case  of  these  two  latter  substances  does  not  differ 
greatly  from  that  of  the  so-called  Ehrlich's  reaction,  and  on 
this  account  the  possibility  of  error  in  interpretation  must 
always  be  borne  in  mind. 

Ehrlich's  diazo-reaction  is  usually  performed  as  follows: 
An  equal  volume  of  a  freshly  prepared  solution  of  diazo- 
benzenesulphonic acid  is  added  to  the  urine  and  the  mixture 
is  then  rendered  alkaline  with  an  excess  of  NH4OH.  An 
orange  color  develops  in  ordinary  cases,  but  in  certain 
urines  there  results  a  red  color  which  may  vary  from  a  car- 
mine to  a  deep  ruby-red;  upon  shaking  the  solution  the 
froth  also  partakes  of  the  color.  Sometimes  a  green  or 
violet  precipitate  will  settle  out  upon  standing. 

The  formation  of  the  fresh  diazobenzenesulphonic  acid  is 
effected  by  the  previous  preparation  of  the  two  following 
solutions : 

1.  1  grm.  sulphanilic  acid  and  50  c.c.  of  cone.  HC1  dis- 
solved in  a  liter  of  water. 

2.  5.0  grm.  sodium  nitrite  dissolved  in  a  liter  of  water. 
Just  previous  >  to  use  these  two  solutions  are  mixed  in 

the  proportion  40 : 1  and  the  mixture  used  as  above. 

The  free  nitrous  acid  which  is  liberated  by  the  action  of 
the  HC1  upon  the  NaN02,  reacts  with  the  sulphanilic  acid 
with  the  formation  of  diazobenzenesulphonic  acid,  according 
to  the  equation 

/NH2  /N^ 

C8H/          +HN02=C6H/ 
XHS03  XSO 


SEDIMENTS. 

UNORGANIZED. 

Separating  from  a  urine  which  is  acid  in  reaction,  the  fol- 
lowing sediments  may  be  present : 

1.  CRYSTALLINE  TYPE.* 

(a)  Uric  Acid, — Color,  golden  brown.  To  what  is  this 
due?  Does  not  dissolve  upon  warming.  Soluble  in  NaOH 
and  re  precipitated  by  HC1.  Responds  to  the  murexide 
test.  Very  characteristic  crystalline  form  under  the  micro- 
scope. 

(6)  Calcium  Oxalate. — Usually  present  mixed  with  uric 
acid.  Colorless.  Dissolves  easily  in  HC1,  but  is  insoluble  in 
acetic  acid.  (See  Triple  Phosphate,  with  which  there  is  the 
possibility  of  confusion.)  Under  the  microscope  the  crystals 
are  transparent,  refractive,  octahedral  (envelope  shape). 

(c)  Bilirubin  and  Hcematoidin. — The  former  crystallizes  in 
golden  or  brown  rhombic  plates  or  needles.     Dissolves  easily 
in  alkalies  and  chloroform  and  gives  Gmelin's  reaction.    The 
latter  is  similar  in  crystalline  form,  but  does  not  dissolve 
in  alkali  and  gives  a  blue  coloration  with  HN03. 

(d)  Cystine. — Under  the  microscope  it  appears  as  super- 
imposed six-sided  plates,  which  are  insoluble  in  acetic  acid, 
but  soluble  in  NEUOH  (differing  from  uric  acid). 

(e)  Tyrosine,   Leucine,   and  Xanthine. — Very  rare.     For 
teste  see  under  these  substances. 

134 


SEDIMENTS.  135 

(/)  Phosphates. — 1.  Magnesium  Phosphate.  Rhombic 
plates,  soluble  in  acetic  acid,  slightly  attacked  by  ammonium 
.carbonate.  2.  Calcium  Phosphate.  Soluble  in  acetic  acid. 
Crystals  wedge-shaped,  seldom  found.  3.  Ammonio-magne- 
sium  Phosphate  (Triple  Phosphate).  These  separate  only 
when  the  reaction  is  weakly  acid  or  amphoteric. 

(g)  Potassium  Sulphate. — Long  colorless  needles,  insoluble 
in  NH4OH  or  acids;  seldom  found. 

2.  AMORPHOUS  TYPE. 

(a)  Uric  Acid  Salts  (Acid  Urates). — Brick-red  or  brown- 
ish-red in  color.  Dissolves  upon  warming  and  gives  the 
murexide  test.  Upon  the  addition  of  a  mineral  acid,  free 
uric  acid  separates  out  in  small  crystalline  form. 

(6)  Calcium  Oxalate. — Dumb-bell  shape.  See  above  for 
detection. 

(c)  Calcium  Sulphate. — Dumb-bell  shape,  insoluble  in  HC1. 

(d)  Fat. — Strongly  refracting  round  drops,  soluble  in  ether. 

Sediments  separating  from  an  alkaline  reacting  urine: 

1.  CRYSTALLINE  TYPE. 

(a)  Triple  Phosphate. — Dissolves  easily  in  acetic  acid; 
unchanged  by  ammonium  carbonate  (see  Magnesium  Phos- 
phate) ;  appears  under  the  microscope  as  large  colorless  prisms 
(coffin-cover  shape).  Upon  warming  gives  off  NH3. 

(6)  Ammonium  Urate. — Dissolves  in  HC1  or  acetic  acid, 
followed  by  the  separation  of  free  uric  acid  crystals  (rhom- 
bic form).  Forms  dark  balls  with  needles  radiating  from  the 
circumference  (chestnut-burs).  Gives  off  NH3  upon  heating 
on  a  platinum  foil. 

(c)  Magnesium  Phosphate. — See  under  Acid  Urine. 


136  LABORATORY  WORK  IN  PHYSIOLOGICAL  CHEMISTRY. 


2.  AMORPHOUS  TYPE. 

(a)  Earthy  Phosphate. — Dissolves  in  acetic  acid  without 
the  development  of  gas. 

(6)  Earthy  Carbonate. — Dissolves  in  acetic  acid  with  effer- 
vescence. 

(c)  Calcium  Carbonate. — Dumb-bell  shape.  Soluble  in 
acetic  acid,  with  an  escape  of  gas.  (Compare  Calcium 
Oxalate.) 

SCHEME  FOR  IDENTIFYING  SEDIMENTS. 

On  heating  the  sediment  on  a  platinum  foil  it 


Does  not  char. 


Does  char. 


Fresh  sediment  treated 
with  HC1 

Fresh  sediment  gives  the 
murexide  test. 

Does  not  effervesce. 

Effervesce,  ^^ 

Sediment  treated  with 
NaOH  gives 

Fresh  sediment  gently 
heated  and  then  treated 
with  HC1 

Ammonia. 

|I 

No  ammonia. 

Srf 

_;     J2. 

q  o 

fl 

Does  not  effervesce. 

Effervesce,  Calcium 

Fresh  sediment  moist- 
ened with  NaOH 

NH3 

la 
P 

NoNH3 

1* 

is 

fj? 

APPENDIX. 


The  methods  for  the  preparation  of  the  solutions,  reagents, 
and  material  of  which  use  is  made  in  the  laboratory  work 
outlined  in  the  preceding  pages,  are  as  follows: 

SOLUTIONS. 

Hydrochloric  acid,  0.2  per  cent;  contains  6  c.c.  of  cone. 
HC1  to  the  liter  of  water. 

Acetic  acid,  20  per  cent  in  water. 

Silver  nitrate,  2  per  cent  in  distilled  water. 

Barium  chloride,  10  per  cent  in  distilled  water. 

Picric  acid,  a  cold  saturated  solution. 

Tannic  acid,  5  per  cent  in  water. 

Phosphotungstic  acid,  10  per  cent  in  water. 

Zinc  acetate,  1  per  cent  in  95  per  cent  alcohol. 

Zinc  chloride,  saturated  water  solution,  diluted  with 
95  per  cent  alcohol  to  a  sp.  gr.  1.20;  filtered. 

Ammonium  oxalate,  a  cold  saturated  solution. 

Alcoholic  potash,  10  per  cent  KOH  in  95  per  cent  alcohol. 

Iodine  solution,  0.5  per  cent  solution  of  potassium  iodide 
saturated  with  iodine. 

Sodium  alcoholate,  sodium  dissolved  in  absolute  alcohol. 

Potassium  permanganate  -TTJ,  3.16  grms.  in  a  liter. 

137 


138  APPENDIX. 

Potassium  mercuric  iodide,  same  as  Tanret's  reagent 
without  the  addition  of  acetic  acid. 

REAGENTS. 

Ammonium  Molybdate. 

50  grms.  molybdic  acid  dissolved  in  200  grms.  10  per  cent 
NH4OH;  pour  this  solution  slowly  into  750  grms.  HNO3 
(sp.  gr.  1.2);  allow  the  mixture  to  stand  for  several  days; 
then  filter. 

Ammoniacal  Silver  Nitrate. 

2.5  per  cent  in  water;  to  this  is  added  NH4OH  until  the 
precipitate  is  completely  dissolved. 

Barfoed's  Reagent. 

4  per  cent  copper  acetate  in  water;  the  solution  made 
.faintly  acid  with  acetic  acid  and  filtered. 

Benedict's  Solution. 

Dissolve  with  heat  173  grams  of  sodium  citrate  and  100 
grams  of  anhydrous  sodium  carbonate  in  about  600  c.c.  of 
water.  Dissolve  17.3  grams  of  cupric  sulphate  in  about  150 
c.c.  of  water.  Mix  the  two  solutions  by  slowly  pouring  the 
cupric  sulphate  into  the  carbonate-citrate  solution.  Make 
the  volume  up  to  1000  c.c. 

Bial's  Reagent. 

500  c.c.  of  30  per  cent  HC1  in  which  are  dissolved  1  grm. 
of  orcinol  and  25  drops  of  a  63  per  cent  solution  of  crystalline 
ferric  chloride. 

Esbactis  Reagent. 

10  grms.  picric  acid  and  20  grms.  citric  acid  dissolved 
in  a  liter  of  water. 


APPENDIX.  139 

Fehling's  Solution. 

A  mixture  in  equal  volumes  of  the  following  solutions: 

1.  Copper    sulphate    solution,    69.28    grms.    crystalline 
CuS04  dissolved  in  water  and  made  up  to  a  liter. 

2.  Alkaline  tartrate  solution,  346  grms.  crystalline   Ro- 
chelle   salts   dissolved  in  350  c.c.  of  water  and  250  grms. 
NaOH  dissolved  in  300  c.c.  of  water;  these  two  mixed  and 
the  volume  made  up  to  a  liter. 

Hammarsten's  Reagent. 

I  volume  HN03  and  19  volumes  HC1  (both  acids  about 
25  per  cent).  This  acid  mixture  should  be  kept  at  least  a 
year.  Then  mix  with  it  four  times  its  volume  of  95  per 
cent  alcohol. 

Hopkins-Cole  Reagent. 

60  grms.  sodium  amalgam  added  to  1  liter  of  a  saturated 
solution  of  oxalic  acid.  After  the  development  of  gas  has 
ended,  filter  and  dilute  with  2  or  3  volumes  of  water. 

Hypobromite  Solution. 

Mix  100  c.c.  of  each  of  the  following  solutions  and  dilute 
with  300  c.c.  of  water. 

1.  125  grms.  bromine  and  125  grms.  sodium  bromide 
dissolved  in  water  and  the  volume  made  up  to  1000  c.c. 
Keep  in  stoppered  bottle. 

2.  A  solution  of  sodium  hydrate  with  a  sp.  gr.  1.250. 

Magnesium  Mixture. 

100  grms.  MgS04  and  200  grms.  NH4C1  dissolved  in  800  c.c. 
of  water;  to  this  add  400  grms.  concentrated  NH4OH. 
Mix  thoroughly  and  keep  in  glass-stoppered  bottle. 


140  APPENDIX. 


Milloris  Reagent. 

To  a  given  amount  of  metallic  mercury  add  twice  its 
weight  of  HN03  (sp.  gr.  1.42);  after  the  evolution  of  gas  has 
ceased,  warm  slightly  and  then  dilute  the  mixture  with  2  vol- 
umes of  water.  Allow  this  to  stand  at  least  24  hours;  then 
filter. 

Morner's  Reagent. 

1  volume  formalin,  45  volumes  distilled  water  and  55 
volumes  concentrated  H2S04;  thoroughly  mixed. 

Nylander's  Reagent. 

2  grms.  bismuth  subnitrate  and  4  grms.  Rochelle  salts 
digested  on  the  water  in  100  c.c.  of  10  per  cent  NaOH;  cool 
and  filter. 

Obermayer's  Reagent. 

1  liter  of  fuming  concentrated  HC1  to  which  has  been 
added  2-4  grms.  ferric  chloride. 

Roberts'  Reagent. 

1  volume  concentrated  HN03  mixed  with  5  volumes  of  a 
saturated  solution  of  MgS04. 

Spiegler's  Reagent. 

8  grms.  mercuric  chloride,  4  grms.  tartaric  acid,  and 
20  grins,  glycerol  dissolved  in  200  c.c.  of  water. 

Tanret's  Reagent  (Potassium-Mercuric-Iodide). 
3.32  grms.  potassium  iodide  dissolved  in  20  c.c.  of  water; 
to  this  add  1.35  grms.  mercuric  chloride  also  dissolved  in 
20  c.c.  of  water;  dilute  the  mixture  to  60  c.c.  and  mix  with 
it  20  c.c.  glacial  acetic  acid. 


APPENDIX.  141 


Uffelmann's  Reagent. 

1  per  cent  solution  of    carbolic  acid  in  water  colored 
faintly  amethyst  with  a  solution  of  ferric  chloride. 

INDICATORS. 

Alizarin. 
1  grm.  dissolved  in  100  c.c.  of  water  and  filtered. 

Boas'  Reagent. 

5  grms.  resorcinol  and  3  grms.  saccharose  dissolved  in 
100  c.c.  95  per  cent  alcohol. 

Congo  Red. 

1  grm.  dissolved  in  90  c.c.  of  water  to  which  is  added 
10  c.c.  of  95  per  cent  alcohol. 

Dimethylaminoazobenzene. 
0.05  grm.  dissolved  in  100  c.c.  95  per  cent  alcohol. 

Gunzburg's  Reagent. 

2  grms.  phloroglucinol  and  1  grm.  vanillin  dissolved  in 
100  c.c.  95  per  cent  alcohol.     (Does  not  keep  well.) 

Phenolphthalein. 
1  grm.  dissolved  in  100  c.c.  95  per  cent  alcohol. 

Tropceolin  00. 
0.05  grm.  dissolved  in  100  c.c.  50  per  cent  alcohol. 


142  APPENDIX. 

MATERIAL. 

Fusion  Mixture. 

5  parts  Na^COa  in  substance  and  1  part  KN03  in  sub- 
stance, intimately  mixed. 

Litmus  Milk. 

Add  a  strong  filtered  solution  of  litmus  to  fresh  milk 
until  the  latter  is  decided  bluish  in  color. 

Neutral  Olive-oil. 

Ordinary  olive-oil  thoroughly  shaken  with  a  10  per  cent 
solution  of  Na^COg;  then  extract  the  mixture  with  ether. 
The  residue  after  the  evaporation  of  the  ether  is  neutral  fat. 

Oxalated  Blood  Plasma. 

As  the  blood  flows  from  an  artery,  allow  it  to  mix  thor- 
oughly in  a  beaker  with  an  equal  volume  of  0.2  per  cent 
ammonium  oxalate  solution. 

Salted  Blood  Plasma. 

Allow  the  blood  flowing  from  an  artery  to  mix  thoroughly 
with  an  equal  volume  of  a  saturated  solution  of  Na2S04  or  10 
per  cent  NaCl.  Place  the  mixture  away  in  a  cold  place  and 
let  it  remain  for  24  hours. 

Pancreatic  Extracts. 

Proteolytically  active:  finely  divided  gland  digested  for 
three  days  with  water  containing  5-10  c.c.  of  chloroform 
to  the  liter,  or  with  a  saturated  solution  of  NaCl. 

Amylolytically  active:  water  or  glycerol  extract  of  the 
gland  previously  allowed  to  stand  exposed  to  the  air  for 


APPENDIX.  143 

24  hours;  or  finely  divided  gland  digested  with  saturated 
solution  of  NaCl;  or  digestion  of  the  finely  comminuted 
gland  at  40°  C.  in  0.7  per  cent  NaCl. 

Lipolytically  active:  slightly  alkaline  watery  infusion  of 
the  gland;  or  an  alkaline  glycerol  extract  of  the  fresh  gland 
(9  parts  glycerol  and  1  part  1  per  cent  Na^COg  solution). 

Rennetically  active:  finely  minced  fresh  gland  digested 
with  4  times  its  weight  of  25  per  cent  alcohol  for  4  to  5  days. 
Then  filter  after  the  addition  of  a  trace  of  acetic  acid. 


INDEX. 


Acetone 131 

Acetic    acid    and    potassium 

ferrocyanide  test 19,  123 

Achroodextrin 44 

Acid  albuminate 54 

Acidic  radicals  in  urine 89 

Acidity  of  urine,  quantitative 

determination  of 105 

Adamkiewicz'  test 18 

Albumin 21 

,  coagulation  of 21 

in  urine 121-124 

Albuminates,  acid  and  alkali.     24 

Albuminoids 27,  28 

Alcohol  as  protein  precipitant     20 

Alizarin  as  indicator 48 

,  preparation  of 141 

Amino  bodies 54 

Ammonia  in  urine 117 

,  quantitative  deter- 
mination of  ....  117 
Ammoniacal    silver    nitrate, 

preparation  of 138 

Ammonium  molybdate,  prep- 
aration of  ....    138 
sulphate  as  pro- 
tein precipitant    20 

urate 135 

Amygdalin 44 

Amylolysis 44,  60 

Amylopsin 56,  60 

Amyloses 1,  7,  8 

Arabinose 2 

Appendix 137 

Bacterium  Micrococcus  urece  .     91 

Barfoed's  test 4 

reagent,     prepara- 
tion of 138 


PAOB 

Basic  radicals  in  urine 90 

Baybenywax,saponification     10 

Benedict's  reagent 138 

test  for  dextrose.    125 

Benzidine  reaction 73,    127 

Bial's  reagent 101,    138 

Bile _ 67 

acids,  conjugate 68 

pigments,  tests  for 69,  70 

salts,  crystallized  (Platt- 

ner's) 69 

Biliary  calculus,  analysis  of . .     70 

Bilirubin 69 

in  sediments 134 

Biliverdin 69 

Biurates 92 

Biuret  test 18 

Blood,  general 72 

pigments 75 

in  urine 127 

,    spectroscopic 
examination 

of 77 

plasma 74 

,  oxalated 74 

,  prepara- 
tion of  142 

,  salted 74 

,  prepara- 
tion of  142 

platelets 75 

Blood  proteins 73 

in  urine 127 

Boas'  reagent,  test  for  HC1 .  .      47 
,  preparation  of.  141 

Boettger's  test 125 

Bone... 36 

,  mineral  constituents .  .     36 

of 36 

145 


146 


INDEX. 


Calcium  carbonate    in    sedi- 
ment      136 

oxalate  in  sediment.  134 
phosphate    in    bone 

ash  .  .  .  . 36 

phosphate  in  milk  . .     83 

in  sediment  135 

sulphate  in  sediment  135 

Cane-sugar 6 

Carbohydrates 1 

in  urine 100 

Carbon,  test  for 2 

Carbonates  in  bone  ash 37 

,  earthy,  in  sediment  136 

Casern   55 

,  pancreatic 61 

Caseinogen,  test  for 82 

Celluloses 7 

Cerebrin 40 

Cerebrosides 38,  40 

Chlorides  in  bone  ash 36 

in  urine 89 

,  quantitative     de- 
termination of  .  106 

Cholesterol  in  bile 70 

in  biliary  calculus     70 
in  nervous  tissue.     41 
Chymosin  (see  Rennin). 
Coagulation  of  proteins  ....      21 
,  method  of  de- 
termination    22 

CO-haemoglobin 79 

Collagen 27 

Color  reactions  for  proteins  .      17 

of  urine 86 

Conductivity,     electrical,    of 

urine 104 

Congo-red  as  indicator 47 

,  preparation  of  ...    141 
Conjugate  sulphates  ....   95,  109 
proteins.  ...   15,  25-27 
Copper   sulphate   as   protein 

precipitant 20 

Creatine  in  muscle 31 

,  transformation  into 

creatinine 31 

Creatinine,  preparation  from 

creatine 31 

in  urine 95 

Cresol,  p- 64,  96 

Cystine  in  urine 129 

in  sediment 134 


PA8B- 

Depression   of  freezing-point 

of  urine 103 

Deuteroproteoses 52 

Dextrin 7 

Dextrose,  tests  for 4 

in  blood 74 

in  urine 125 

Diacetic  acid  in  urine 131 

Dialysis  experiment 23 

Digestion,  gastric 46 

,  peptic 51 

,  pancreatic 56 

,  tryptic 57 

Dimethylaminoazobenzene  as 

indicator  47 

,  preparation  of  141 

Disaccharides 1,  5,  7" 

Edestin 23 

Ehrlich's  diazo  reaction .  132,  134 
Electrical  conductivity  of 

urine 104 

Emulsification  of  fats 13 

Enzymes,  preparation  of.  ...  142 

Erythrodextrin 44 

Esbach's  reagent,  preparation 

of 138 

Fat 9 

,  emulsification  of -   13 

in  milk 83 

in  sediment 135 

,  saponification  of  .   ...   10,  11 
Fatty    acids,    derivatives    in 

urine 130 

in  nervous  tissue  38 

,  perparation  of.  11 

,  reactions  for .  .  12' 

Fehling's  test 4 

solution,      prepara- 
tion of 139 

Fermentation  of  dextrose  ...  5 

of  saccharose.  .  7 

Fibrin 74 

Form  elements  in  blood 75 

Freezing-point,  depression  of, 

in  urine 103: 

Fusion  mixture,preparation  of  142 

Galactose $ 

Garrod's  method  for  haemato- 

porphyrin 128 


INDEX. 


147 


Gastric  digestion 46 

juice 46 

Gelatin,  tests  for 28 

Globulin  in  urine 121 

,  tests  for 23 

Glucose  (see  Dextrose). 

Glycerol 14 

Glycocholic  acid 68 

Glycogen  in  muscle 34 

,  tests  frr: ,  „ 35 

Gl y  coprotein 25 

Glycoproteose 53 

Glycuronic  acid 102 

Gmelin's  test  for  bile  pigments    70 
Guaiac  reaction  for  blood  ...     73 

for  milk 81 

Gum  arabic 2 

cherry 2 

Gunnings'     modification     of 

Legal's  test  for  acetone.  .  .    132 
Giinzburg's  reagent,  test  'for 

HC1  .  .     47 
,  prepara- 
tion of  141 

Hsematin 75 

in  urine 125 

,  reduced 78 

Haematoidin  in  sediment 134 

Haematoporphyrin 78 

in  urine  .  .  1 28 

Hsematuria 128 

Ha?min  crystals,  test  for  blood    76 

Haemochromogen 78 

Haemoglobin 75,  77 

carbon  monoxide 
(see  CO-hemoglobin). 

Haemoglobinuria 127 

reagent,  prep- 
aration of.   139 
Hammarsten's  test   for   bile 

pigments     70 
for  indican     97 

Haycraft's  test 127 

Heller's  test  for  protein 19 

for  oxyhaemoglo- 

bin 127 

Heteroproteose 52 

Hexoses 1 

Hippuric  acid 98 

Hopkins-Cole  reagent,  prepa- 
ration of . .          139 


Hopkin's-Fplin    method    for 

uric  acid 115 

Hilfner's  method  for  urea  ...  114 

Huppert's  test  for  bile  pig- 
ments   70 

Hydrochinol,    conjugate     in 

urine 96 

Hydrochloric  acid  in  gastric 

juice 46 

Hydrogen,  test  for 2 

Hypobromite  solution,  prepa-  . 

ration  of 139 

Hypoxanthine 32 

Indican 97 

Indicators 47 

Indigo-carmin  test  for  lactose  84 
Indole  in  intestinal  putrefac- 
tion     62-64 

conjugate  in  urine  ...  96 

Inorganic  compounds  in  urine  88 

Intestinal  putrefaction 62 

Inversion 6 

Invertin 6 

Iron  in  bone  ash 37 

in  protein 17 

,  qualitative  test  for  ....  17 

Jaffe's  test  for  creatine 37 

for  indican 97 

Keratin,  test  for 28 

Lactic  acid,  test  for,  in  stom- 
ach contents 47,  48 

Lactose 5 

in  milk 84 

Lard,  saponification  of 11 

Lead  oleate 12 

Lecithins 39 

Lecithoproteins 15 

Legal's  reaction  for  acetone  .  132 

for  indole  ...  64 

Leucine,  tests  for 59 

in  sediment 134 

Levulose 3 

Lieben's  test  for  acetone  ....  131 
Liebermann's  test  for  choles- 
terol    42 

Lipoids  of  nervous  tissue ....  38 

Lipolysis 60 

Litmus  milk 60 

,  preparation  of .  142 


148 


IXDEX. 


9A.GE 

Magnesium  in  bone  ash 37 

mixture,  prepara- 
tion of 139 

Magnesium  sulphate  as  pro- 
tein precipitant 20 

Maltodextrin 44 

Maltose 5,  44 

Metacasein  reaction 61 

Methsemoglobin 78 

Milk,  general 80 

,   qualitative    separation 

of  the  constituents  of.  81 
Millon's  reagent,  preparation 

of 140 

test  for  proteins ....  18 

Mineral  constituents  of  bone .  36 

Monosaccharides 1,  3,  5 

Moore's  test  for  hexoses  ....  4 
Morner's  reagent,  preparation 

of 140 

test 59 

Mucins  in  bile 67 

in  saliva 43 

,  tests  for 25 

Murexide  test 94 

Muscular  tissue 29 

,    nitrogenous 

extractives  of  30 
,   non-nitroge- 
n  o  u  s  e  x  - 

tractives  of  34 

,  proteins  of .  .  29 

Myogen 29 

Myosin 30 


Nervous  tissue 38 

,  cerebrin  of  .  .  40 
,  cholesterol  of.  38,  41 
,  fatty  acids  of.  38 
,  lecithins  of  .  .  39 
,  lipoidsof.  .  .  .  38 
,  neurokeratin 

of 38 

,  proteins  of  .  .     38 

Neurokeratin 38 

Neutral  fats  (see  Fats). 

Neutral  olive-oil 13 

,  preparation  of  142 
Nitrogen,  qualitative  tests  for     15 
,  total,    quantitative 
determination     of 
(Kjeldahl)..    112-114 


Nitrogenous  extractives 

of  muscular  tissue  30 

of  nervous  tissue  38 

Nucleins , 26 

,  tests  for 27 

Nucleoproteins 26 

Nylander's  reagent,  prepara- 
tion of 140 

test 4,  125 


Obermeyer's  reagent,  prepar- 

tion  of 140 

test  for  indican .  97 

Odor  of  the  urine 86 

Oleate,  lead 12 

Oleicacid 12 

Olive-oil,  neutral 13 

,  preparation  of  ....  141 

Orcinol  as  test  for  pentoses. .  .  3 

Ossein  of  bone 27,  36 

Oxalic  acid 97 

preparation  from  urine  98 

/J-oxybutyric  acid  in  urine ...  130 

OxyhaBmoglobin 75,  127 

Palmitic  acid 11 

Pancreatic  digestion 56 

extracts,  prepara- 
tion of 138 

rennin 56,  61 

Pentosanes 2 

Pentoses,  tests  for 2 

in  urine 101 

Pepsin 49-51 

Pepsinogen 50 

Peptic  proteolysis 51 

Peptones 51,  53,  54 

in  intestinal  putre- 
faction       62 

Pettenkofer's    test    for    bile 

acids 68 

Phenol,  conjugated  in  urine  .      96 
Phenolphthalein  as  indicator.     48 
,  preparation 

of 140 

Phenylhydrazin  reaction.  .  5,  126 
Phloroglucinol,  as  test  for  pen- 

tose 3 

Phosphates,  earthy,  in  urine  .111 

in  sediment 135 

in  urine .   109 


INDEX. 


149 


PAGE 

Phosphates,  quantitative  de- 
termination of 109,  110 

Phosphoric  acid  in  bone  ash .  .  36 

Phosphorus  in  protein 17 

,  organic,  in  urine.  110 
,  qualitative  test 

for,  in  urine ...  Ill 

Phosphoprotein 26,  122 

Phosphotungstic  acid,  as  pro- 
tein precipitant 20 

Picric  acid  as  protein  precipi- 
tant    20 

Pigments,  biliary 69 

,  blood,  in  urine  ...  127 

,  urinary 99 

Piria's  test  for  tyrosine 59 

Plattner's  crystallized  bile  .  .  69 

Polarization 5 

of  urine 126 

Polysaccharides 1,  7,  8 

Potassium  mercuric  iodide  . .  20 
sulphate    in    sedi- 
ment    135 

sulphocyanide     in 

saliva 43 

Precipitation  reactions 19 

Protagon 38 

Proteins 15 

,  coagulation  of.  .  .  21,  22 
,  compound  .  .    15,  25,  27 

in  blood  serum 73 

in  milk 83 

in  nervous  tissue  ...  29 

in  urine 121 

,  quantitative  deter- 
mination of 118 

Proteoses 51,  124 

in  intestinal  putre- 
faction    62 

in  urine 124 

Protoproteose 52 

Ptyalin 44 

Purine  bases  in  muscle 32 

in  urine 94 

,  method  of  sep- 
aration    33 

Putrefaction  experiment  and 

separation 63 

Pyrocatechinol,  conjugated  in 


Quadiurates 92 


PAGE 

Reaction  of  the  urine 86 

Reagents,  preparation  of  ...    138 

Reduced  hsematin 78 

hemoglobin 77 

Rennin 55 

Rhamnose 101 

Robert's  modification  of  Hel- 
ler's test 123 

reagent,  preparation 

of 140 

Rosenbach's  modification  of 
Gmelin's  test 126 

Saccharose 1,  5-7 

Salivary  digestion 43 

Salkowski's  test  for  choleste- 
rol       42 

Saponification    of    bayberry 

wax 10 

of  lard 11 

Scherer's  test  for  leucine  ....     59 
Schlosing's  method  for  ammo- 
nia     117 

Sediments  in  acid  reaction  .  .  134 
in  alkaline  reaction  135 
,  scheme  for  identi- 
fying    136 

unorganized 134 

Serum  albumin 73 

globulin 73 

Skatole-carbonic  acid  ....   62,  65 
conjugated  in  urine . .     96 
in    intestinal    putre- 
faction    62-64 

Soaps 12 

Sodium  alcoholate 83 

chloride     in     blood- 

cerum 74 

Solutions,  preparation  of ....  137 
Smith's  test  for  bile  pigments  70 
Specific  gravity  of  the  urine .  .  87 
Spectroscopic  examination  of 

the  blood 77 

Spiegler's   reagent,    prepara- 
tion of 140 

test 124 

Starches 7 

Starch  paste 8 

Steapsin 56,  60 

Stokes'  reagent 77 

Struve's  test  for  oxyhaemoglo- 
bin..  .   128 


150 


INDEX. 


Sugar  in  blood 74 

Sulphates,  conjugate,  in  urine  109 
,  quantitative  deter- 
mination of  ....  107 

,  total 108 

Sulphur  in  urine  neutral  ....  89 

,  lead-blackening ....  15 

,  oxidized 16 

,  qualitative  test  for  .  15 

Synproteose 53 

Tannic  acid,  as  protein  pre- 
cipitant   i 19 

Tanret's  reagent,  preparation 

of 140 

test 124 

Taurocholic  acid 68 

Teichmann's  crystals,  test  for 

blood 75 

Tetroses 1 

Thioproteose 53 

Total  solids  in  urine 88 

Trichloracetic  acid,   test  for 

proteins 20,  123 

Trioses 1 

Trommer's  test  for  hexoses . .  4 

Tropseolin  OO  as  indicator. . .  47 

,  preparation  of  141 

Trypsin 56,  57 

Trypsinogen 57 

Tryptic  proteolysis 57 

Tryptophane 62,  66 

Tyrosine,  test  for 58 

in  sediment 134 

Uffelman's    reagent,test    for 

lactic  acid  49 
,  preparation 

of 141 

Urates 92 

,  ammonium,  in  sedi- 
ments    135 


PASS 

Urea 91 

,  preparation  of 91 

,  quantitative     determi- 
nation of 114 

Uric  acid 92 

,  preparation  of 93 

,  quantitative  deter- 
mination    115 

Uric  acid  in  sediment 134 

,  tests  for 93 

Urine 85 

,  acidity  of 105 

,  quanitative 
determina- 
tion of .  86 

,  color  of 86 

,  odor  of 86 

,  physical  properties  of .  86 
,  quantitative  deter- 
mination of. 103,  104 

,  reaction  of 86 

,  sediments  in 88,  134 

,  specific  gravity  of  ....  87 
,  tests  for  normal  con- 
stituents of 88 

,  total  solids  of 88 

,  transparency  of 86 

,  volume  of 87 

Urinary  pigments 99 

Urobilin 99 

,  test  for 100 

Urochrome 99 

Volume  of  the  urine 87 

Water,  test  for  hardness  ....     12 
Weyl's  test  for  creatinine  ...     32 

Xanthine 32 

in  sediments 134 

Xanthoproteic  reaction 12 

Xylose 8 


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